Method of detecting progression of a neurodementing disease

ABSTRACT

Isolated, monoclonal, human, anti-β-amyloid antibodies are provided which bind to dimeric forms of Ab with higher affinity than to monomeric forms of Ab and when bound to an Aβ polypeptide comprising Aβ(21-37) shield Aβ(21-37) from proteolytic digestion. The antibodies were shown to inhibit fibril formation and reduce plaque size in vivo and to not bind brain vessel walls. Accordingly, the antibodies are useful in human and veterinary medicine for the treatment and prophylaxis of Alzheimer&#39;s disease and other neurodementing diseases. Methods of detecting or measuring the progression of a neurodementing disease also are provided.

INFORMATION ON RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/884,513, filed Jan. 11, 2007, and to U.S. Provisional Application No.60/884,526 filed Jan. 11, 2007, and to U.S. Provisional Application No.60/981,667, filed Oct. 22, 2007, and to U.S. Provisional Application No.60/981,675, filed Oct. 22, 2007, and to European Patent Application No.07000507.9, filed Jan. 11, 2007, and to European Patent Application No.07000521.0, filed Jan. 11, 2007, and to European Patent Application No.07119002.9, filed Oct. 22, 2007, and to European Patent Application No.07119026.8, filed Oct. 22, 2007, all of which are hereby incorporated byreference in their entireties.

BACKGROUND

Alzheimer's disease (AD), the most common form of dementia among elderlypopulation (prevalence: 1000/100,000; >65 years), represents the fourthleading cause of death in the developed world. Cortical atrophy,neuronal loss, region-specific amyloid deposition, neuritic plaques, andneurofibrillary tangles are key neuropathological features in the ADbrain. These alterations are thought to be linked to cognitive declinewhich clinically defines AD. Within these markers, neuritic plaques areamyloid immunoreactive, thioflavin positive, and accompanied byastrogliosis, microgliosis, cytoskeletal changes, and synaptic loss. Thedegree of neuritic degeneration within plaques correlates with clinicalparameters of dementia. Neuritic plaques are spherical, multicellularlesions that are usually found in moderate to large numbers in limbicstructures and associated neocortices of the AD brain. These plaques arecomprised of extracellular deposits of amyloid-βpeptide(s) (Aβ) thatinclude abundant amyloid fibrils intermixed with non-fibrillary forms ofthe peptide. Such plaques also contain variable numbers of activatedmicroglia that are often situated very near the fibrillar amyloid core,as well as reactive astrocytes surrounding the core.

The major constituent of the neuritic plaque, β-amyloid polypeptide(Aβ), arises from a larger precursor protein, the amyloid precursorprotein (APP) (Kang, et al., 1987; Tanzi, et al., 1987). Aβ is producedby normal cells and can be detected as a circulating peptide in theplasma and cerebrospinal fluid (CSF) of healthy humans. Although thephysiological role of the amyloid precursor protein (APP) in the brainis not well understood, missense mutations in APP confer autosomaldominant inheritance of AD (FAD), and shed light on potentiallyimportant pathogenic mechanism(s). The accumulation of Aβ, a 39-42 aminoacid proteolytic product of APP, in neuritic plaques, structures whichat autopsy fulfill the neuropathological criteria for a definitivediagnosis of AD, is thought to be causative for disease progression. Amajor Aβ cleavage product of APP is the Aβ(1-42) polypeptide, but Aβpeptides shorter at the C-terminus (39 to 41) are also produced by theproteolytic (γ-secretase) cleavage in the membrane. The N-terminal partof Aβ(1-42) is localized in the extracellular region of APP, and themajor C-terminal part of the Aβ peptide is contained within thetransmembrane domain.

Missense mutations, in APP associated with FAD, occur in proximity tothe Aβ domain and result in an increase in the production of the 4 kDaAβ peptide. In Aβ, it has been postulated that increased synthesisand/or a decreased clearance of Aβ may lead to amyloid plaque depositionand subsequently to the neuropathological changes associated with thedisease. In vitro studies, using synthetic Aβ peptide(s), have shownthat neurotoxicity is dependent on Aβ being fibrillar and predominantlyin a β-pleated sheet conformation.

The accumulation of extracellular plaques containing the neurotoxicamyloid peptide fragment (Aβ) of β-amyloid precursor protein (APP), asthe major product, is one of the characteristics of Alzheimer's disease(AD). Although APP has been recognized as a key molecule for AD, themolecular (patho-) physiological degradation and proteolytic pathways ofAPP, and cellular interactions and biochemical fate of Aβ peptide(s) arestill unclear. Despite the lack of details on degradation pathways andcellular transport for the formation and deposition of Aβ-derivedplaques, recent studies towards the development of immunisation methodsof AD based on therapeutically active antibodies produced from Aβ(1-42)have yielded initial success in transgenic mouse models of Alzheimer'sdisease. Several reports have demonstrated that antibodies generated byimmunization with Aβ(1-42) are capable of inhibiting the formation ofAβ-plaques by disaggregating Aβ-fibrils, and improve the impairments inthe spatial memory of mice. The transgenic APPV717F mouse (TG mouse) isa well characterized model of AD-like plaque pathology with age- andregion-dependent deposits of Aβ(1-40) and Aβ(1-42) (Games, et al.,1995). Recently, Schenk et al. and others investigated alterations inthe deposition of AD in APPV717F TG mouse following immunization withpre-aggregated Aβ(1-42) or administration of antibodies against Aβ(Bard,et al., 2000; Schenk, et al., 1999). Both immunization andadministration of AD antibodies significantly attenuated amyloid plaquedeposition, neuritic dystrophy, and astrogliosis. In these studies,increased titers of mouse anti-human Aβ-antibodies were necessary forthe observed reduction in plaque burden. These findings raise thepossibility that formation and clearance of an Aβ-antigen: antibodycomplex may decrease brain Aβ deposition either following antibodygeneration within the central nervous system or by peripheral antibodytransport across the blood-brain-barrier (BBB). Furthermore, passiveimmunization appears to reduce brain Aβ burden by altering Aβequilibrium between the CNS and plasma (DeMattos, et al., 2001).Remarkably, active or passive immunization significantly reversesbehavioral and memory impairment in APPV717F mouse or other APPtransgenic mice (Dodart, et al., 2002; Janus, et al., 2000; Morgan, etal., 2000). These results suggest that immunization may prevent memorydeficits possibly by altering a soluble pool of Aβ. Thus, treatment ofAD patients with active or passive immunization is one of severalemerging therapeutic approaches targeting the production, clearance, andaggregation of the AD peptide.

Based on these results, a clinical trial using an active immunizationprocedure [Aβ(1-42) peptide and/or preaggregates thereof; adjuvant:QS21] was initiated for treatment of patients with established AD.Unfortunately, severe side-effects developed (“meningoencephalitis”) andthe clinical trial was stopped. A subgroup of AD patients (n=30) treatedwith active immunization in this clinical trial, was analyzed (Hock, etal., 2002; Hock, et al., 2003). The authors demonstrated that (i)immunization induces the production of antibodies against Aβ(1-42) and(ii) in patients where a production of antibodies was observable, thecognitive decline was significantly reduced in comparison to theuntreated control group. The authors concluded that immunization may bea therapeutic option for AD.

Recent studies elucidated in more detail the recognition properties ofantibodies produced upon immunization with Aβ(1-42). This work resultedin the identification of a specific Aβ-epitope recognized by theantibodies generated in transgenic AD mice (McLaurin et al., 2002;Przybylski et al., 2003). These results have been obtained by usingselective proteolytic excision technologies (Epitope-Excision) incombination with high resolution mass spectrometry (FTICR-MS) asbioanalytical tools of high sensitivity and specificity for theidentification of antigen epitopes (Macht et al 1996; Suckau et al 1992;Macht et al. 2004; see FIGS. 1, 2)). Using mass spectrometric epitopeexcision of the immobilized Aβ-antigen-immune complex, the epitope wasidentified to consist of the residues (4-10) (FRHDSGY) of Aβ(1-42). Theselectivity of this recognition structure was ascertained by elucidationof the identical epitope from AD plaques, Aβ(1-42) extracts fromAβ-protofibrils, chemically synthesised Aβ(1-42), and other(Aβ-independent) polypeptides comprising the N-terminal Aβ sequence(Przybylski et al. 2003).

Naturally occurring anti-Aβ autoantibodies (Aβ-autoantibodies) wereidentified by Du et al. in both the blood and the CSF from non-immunizedhumans (Du, et al., 2001). These antibodies specifically recognize humanAβ as has been shown by immunoprecipitation (Du, et al., 2001) andELISA. Furthermore, the antibodies readily recognize synthetic Aβ(1-40)as well as human Aβ deposited in the brain of PDAPP transgenic mice. Inaddition, fibrillation/oligomerization and neurotoxicity of Aβ-peptideswere reduced in the presence of Aβ-autoantibodies (Du, et al., 2003).

Furthermore, it has been investigated whether there is a difference ofthe Aβ-autoantibody concentration in patients with Alzheimer's diseasecompared to controls. Interestingly, a significant difference among thetwo groups was found, resulting in a substantially decreased titer(approximately 15-20-fold) of antibodies against Aβ in patients withAlzheimer's disease. These results have been confirmed recently by othergroups (Weksler et al., 2004). Antibodies against Aβ can also bedetected in commercially available intravenous IgG preparations (IVIgG).The treatment of patients with different neurological diseases withthese intravenous immunoglobulin preparations led to the reduction of Aβconcentration in the CSF (Dodel, et al., 2002). The substantial effectof the Aβ-autoantibodies in preventing, and protecting against Aβ-plaquedeposition was also established in young (4 months) APP-transgenic(TgCRND8) mice. Additionally, in a pilot trial with 5 patients with AD,utilizing IVIgG, total Aβ was reduced significantly in the CSF andincreased in the serum upon delivery of IVIgG (Dodel, et al., 2004). Inthe five investigated patients no cognitive deterioration was observedduring the six months observation period. These results have beenconfirmed by a recent pilot study involving 8 AD patients, who weretreated with IVIgG (Relkin et al., 2006)

However, administering IVIgG to a patient with AD is not convenient andassociated with high costs, as the fraction of therapeutic Aβautoantibodies is low. The vast majority of IgG in this preparation isnot Aβ specific and may result in undesirable effects. Furthermore, thesources for IVIgG are limited, which is an unacceptable disadvantage inview of the prevalence of patients with Alzheimer's disease.

Methods of detecting and monitoring the progression AD and otherneurodementing diseases similarly are inadequate. Current AD diagnosticsfall into three groups: (i) determinations for genetic risk factors ormutations (mainly for FAD cases, but not for sporadic AD diagnostics);(ii) neuroimaging methods; and (iii) diagnostics based onbiochemical/biological markers. Present work on the development ofdiagnostic procedures based on biomarkers have been mainly focused onCSF, which has the principal disadvantage that such methods requireelaborate, invasive material. A major problem associated withbrain-derived biomarkers is that clinically examined controls often alsoinclude subjects with preclinical AD pathology. Further, currentavailable biomarkers have the major disadvantage of low specificity.Similar disadvantages have been noted for a series of proteins expressedin the frontal cortex, identified by brain proteomics approaches, aspotential brain biomarkers arising from presumed alterations of bloodbrain barrier in AD.

Studies on biomarkers in plasma and serum have been performed mainlywith determinations of SP (senile plaques) and NFT (neurofibrillarytangles) components, e.g. the Aβ peptides Aβ(1-40) (SEQ ID NO: 1) andAβ(1-42) (found with elevated levels) and hyperphosphorylatedTau-protein. However the specificity of Aβ determinations, andapplication for early and differential diagnostics has been considereduncertain, the same is the case for protein Tau determinations which hasbeen described as a marker of already progressing neurodegeneration.

There exists, therefore, a need for improved methods of treating anddetecting neurodementing diseases such as AD.

SUMMARY

In one aspect, isolated, monoclonal, human, anti-β-amyloid antibodiesare provided that comprise more than one amino acid sequence selectedfrom at least two consensus amino acid sequences of the group consistingof SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10and SEQ ID NO: 11, wherein each of said more than one amino acidsequence is from a different SEQ ID NO, wherein said antibody binds todimeric forms of Aβ with higher affinity than to monomeric forms of Aβ.

In one embodiment, the antibody comprises more than two amino acidsequences selected from at least three consensus amino acid sequences ofthe group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, wherein each of said more thantwo amino acid sequences is from a different SEQ ID NO. In another, theantibody comprises more than three amino acid sequences selected from atleast four consensus amino acid sequences of the group consisting of SEQID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 andSEQ ID NO: 11, wherein each of said more than three amino acid sequencesis from a different SEQ ID NO. In still another embodiment, the antibodycomprises more than four amino acid sequences selected from at leastfive consensus amino acid sequences of the group consisting of SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQID NO: 11, wherein each of said more than four amino acid sequences isfrom a different SEQ ID NO. In another embodiment, the antibodycomprises an amino acid sequence from each of the consensus amino acidsequences of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQID NO: 10 and SEQ ID NO: 11.

In one embodiment, the antibody comprises the amino acid sequences ofSEQ ID NO: 33, SEQ ID NO: 41; SEQ ID NO: 45 as specific light chain CDRsCDR1, CDR2 and CDR3 respectively and SEQ ID NO: 15; SEQ ID NO: 23 andSEQ ID NO: 29 as specific heavy chain CDRs CDR1, CDR2 and CDR3respectively, while in another the antibody comprises the amino acidsequences of SEQ ID NO: 53 and SEQ ID NO: 60. In other embodiments, theantibody comprises the amino acid sequences of SEQ ID NO: 145 and SEQ IDNO: 60, SEQ ID NO: 51 and SEQ ID NO: 60, SEQ ID NO: 52 and SEQ ID NO:60, SEQ ID NO: 146 and SEQ ID NO: 60, SEQ ID NO: 53 and SEQ ID NO: 148,SEQ ID NO: 55 and SEQ ID NO: 61, or SEQ ID NO: 145 and SEQ ID NO: 62.The antibody also can comprise the CDRs from the amino acid sequences ofSEQ ID NO: 145 and SEQ ID NO: 60, SEQ ID NO: 51 and SEQ ID NO: 60, SEQID NO: 52 and SEQ ID NO: 60, SEQ ID NO: 146 and SEQ ID NO: 60, SEQ IDNO: 53 and SEQ ID NO: 148, SEQ ID NO: 55 and SEQ ID NO: 61, or SEQ IDNO: 145 and SEQ ID NO: 62.

In another aspect the antibody binds to a peptide comprising Aβ(21-37),while in another the antibody shields residues of Aβ(21-37) fromproteolytic digestion when being bound to an Aβ polypeptide comprisingAβ(21-37). In another, the antibody binds specifically to Aβ(12-40) orAβ(20-37) when such antibody is coupled to NHS-activated 6-aminohexanoicacid-coupled sepharose, but does not bind specifically to Aβ(17-28),Aβ(25-35) or Aβ(31-40).

The inventive anti-β-amyloid antibodies also can be formulated aspharmaceutical compositions.

In another aspect, methods of preventing or treating a neurodementingdisease in a patient comprise administering to the patient atherapeutically acceptable amount of the inventive anti-β-amyloidantibodies. Such methods can be used in preventing or treatingneurodementing diseases selected from the group consisting ofAlzheimer's disease, Down's syndrome, dementia with Lewy bodies,fronto-temporal dementia, cerebral amyloid angiopathy and amyloidoses.In a preferred embodiment, the neurodementing disease is Alzheimer'sdisease.

In another embodiment, the inventive anti-β-amyloid antibodies can beused for the manufacture of a medicament in order to treat aneurodementing disease or to slow or prevent the progression of aneurodementing disease.

In another aspect, methods of detecting or measuring the progression ofa neurodementing disease in a patient are provided that comprise (A)measuring in a sample from said patient an antibody titer against afirst Aβ peptide, wherein the first Aβ peptide comprises at least thesequence according to Aβ(30-37) and at most the sequence according toAβ(12-40); (B) measuring in a sample from said patient an antibody titeragainst a second Aβ peptide wherein the second Aβ peptide comprises atleast the sequence according to Aβ(4-10) and at most the sequenceaccording to Aβ(1-20); and (C) comparing the titers from steps (A) and(B). In some embodiments, the first Aβ peptide comprises at least thesequence according to Aβ(21-37). In another embodiment, the methodsfurther comprise comparing the patient titers with titers determined forhealthy donors and AD patients whereby a higher titer against the firstAβ peptide correlates with a lower risk of development and/orprogression of Alzheimer's disease. The methods also can comprisecomparing the patient titers with titers determined for healthy donorsand AD patients whereby a higher titer against the first Aβ peptide,relative to the titer against the second Aβ peptide correlates with alower risk of development and/or progression of Alzheimer's disease.Alternatively, the methods can comprise comparing the patient titerswith titers determined for healthy donors and AD patients whereby ahigher titer against the second Aβ peptide correlates with a higher riskof development and/or progression of Alzheimer's disease. In anotherembodiment, the methods further comprise comparing the patient titerswith titers determined for healthy donors and AD patients whereby ahigher titer against the second Aβ peptide, relative to the titeragainst the first Aβ peptide, correlates with a higher risk ofdevelopment and/or progression of Alzheimer's disease.

Methods of detecting or measuring the progression of a neurodementingdisease in a patient also are provided that comprise A) obtaining afirst sample from said patient at a given time point; B) obtaining asecond sample from said patient at later time point; C) measuring insaid first and second samples the antibody titer against an epitopecomprising at least Aβ(30-37) and at most Aβ(12-40); and D) comparingthe titers of said first and second samples. Other such methods compriseA) obtaining a first sample from said patient at a given time point; B)obtaining a second sample from said patient at later time point; C)measuring in said first and second samples the antibody titer against anepitope comprising at least Aβ(4-10) and at most Aβ(1-20); and D)comparing the titers of said first and second samples. In otherembodiments, such methods comprise A) obtaining a first sample from saidpatient at a given time point; B) obtaining a second sample from saidpatient at later time point; C) measuring in said first and secondsamples the antibody titer against an epitope comprising Aβ(30-37); andD) comparing the titers of said first and second samples.

In another aspect, there is provided a kit comprising (A) a first Aβpeptide comprising at least the sequence according to Aβ(30-37) and atmost the sequence according to Aβ(12-40), and (B) a second Aβ peptidewherein the second Aβ peptide comprising at least the sequence accordingto Aβ(4-10) and at most the sequence according to Aβ(1-20).

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these figures in combination with the detailed description ofspecific embodiments presented herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Principle of epitope-excision and epitope extraction for massspectrometric epitope identification. Antibody immunoglobulin withnative, disulfide-bonding is generally highly resistant to proteolyticdigestion by endoproteases (e.g., trypsin, chymotrypsin, AspN-protease),and the epitope region of antigen polypeptides comprising theepitope-paratope interaction structure is generally protected fromproteolytic degradation in the immune complex, while the free nonbindingregions are amenable to digestion. Thus, the epitope sequence remainingbound to the antibody after proteolytic removal and washing awaynonbinding structures is then dissociated from the antibody andidentified by mass spectrometry. Both electrospray-ionization (ESI) andmatrix-assisted laser desorption-ionization (MALDI) have been founduseful mass spectrometric methods, and have been applied successfullyfor epitope identifications. “TFA” means trifluoroacetic acid.“MALDI-MS” stands for matrix-assisted laser desorption-ionisation massspectrometry.

FIG. 2: Mass spectrometric identification of proteolytic peptidefragments of free soluble Aβ peptide. Without the complexation byantibody binding, digestion of Aβ(1-40) by trypsin leads to formation ofall peptide fragments expected according to the proteolytic cleavagespecificity (Aβ(1-5), Aβ(6-16), Aβ(17-28), Aβ(17-40), Aβ(1-16)). Massspectrometric analysis is performed by high resolutionMALDI-Fouriertransform-ion cyclotron resonance (MALDI-FTICR-MS), whichprovides spectra at approximately 100,000 mass resolution with completeisotope resolution of ions and mass determination accuracies oftypically 1-5 ppm. All FTICR-MS spectra were obtained with a Bruker(Bruker Daltonik, Bremen, Germany) Apex II 7T FT-ICR mass spectrometerequipped with an Apollo II electrospray/nanoelectrospray multiportionsource and an external Scout 100 fully-automated X-Y target stage MALDIsource with pulsed collision gas. The pulsed nitrogen laser is operatedat 337 nm. Ions generated by laser shots were accumulated in thehexapole for 0.5-1 sec at 15 V and extracted at −7 V into the analyzercell. A 100 mg/ml solution of 2,5-dihydroxybenzoic acid (DHB, Aldrich,Germany) in acetonitrile: 0.1% TFA in water (2:1) was used as thematrix. 0.5 μl of sample solution were mixed on the stainless-steelMALDI sample target and allowed to dry. Typical ESI conditions were ˜2kV needle voltage and 100 nA spray current. Ions were accumulated in ahexapole for 2 sec and then transferred into the cylindrical ICR cell.“ppm” stands for parts per million. “m/z” indicates themass-to-charge-ratio.

FIG. 3: Epitope Identification of Amyloid Plaque Specific Antibody byMALDI-FTICR-MS: Mass spectrometric identification of N-terminalAβ-epitope recognized by plaque-specific antibody produced upon activeimmunization of transgenic mice with Aβ(1-42) or Aβ(1-42)-derivedaggregates. The immobilized, purified antibody was incubated withAβ(1-40), Aβ(1-42), and the immune complex subjected to epitope excisionby proteases trypsin, chymotrypsin, Glu-C protease and Asp-N-protease.The left spectrum shows the fragment, Aβ(1-16) remaining bound aftertrypsin digestion, the right spectrum shows Aβ(1-11) after epitopeexcision using Glu-C-protease. Black small arrows shown in the Aβsequence shown denote cleavages identified by epitope excision, fat greyarrows denote substrate cleavage sites on Aβ that were found shieldedupon antibody binding. Identical Aβ(4-10) epitope sequences wereidentified with soluble Aβ-plaques and -protofibrils bound as antigens,and from a mouse anti-Aβ(1-16) peptide monoclonal antibody(Bachem-Peninsula Laboratories, San Francisco).

FIG. 4: Toxicity of Aβ-oligomers for human neuroblastoma cells (SH-Sy5y)in absence or presence of anti-Aβ(21-37) autoantibody as described inExample 4. OD: optical density.

FIG. 5: 1D-Gel electrophoretic separation of polyclonal plaque-specificantibodies from an AD patient (AD77), isolated by Aβ(4-10)epitope-specific affinity chromatography as described in Example 1. KDa:Molecular weight in kilodalton. IgG: Immunoglobulin G. DTT:Dithiothreitol.

FIG. 6: Identification of Aβ(21-37) as the epitope recognized by humananti-Aβ(21-37)-autoantibodies by epitope excision-mass spectrometry. Theupper graph shows the sequence of Aβ(1-40), with cleavages by differentproteases indicated by black arrows. Peptide fragments denoted by solidblack arrows above the Aβ-sequence were identified after epitopeexcision using pronase; peptide fragments denoted by black dotted arrowsunderneath the Aβ sequence were found by epitope excision using trypsinand Glu-C-protease (R5, E11, K16); note that Arg-5 is completelyshielded in the immune complex with the plaque-specific antibody, whilecompletely amenable to cleavage in the immune complex with theanti-Aβ(21-37)-autoantibody. Cleavage positions, observed in free Aβ,indicated by broken arrows were found shielded after binding ofanti-Aβ(21-37)-autoantibody (Glu-C: E22, D23; trypsin: K28). TheMALDI-MS analysis upon partial digestion (2 hrs) with pronase is shownhere for illustration.

FIG. 7: Isolation of “plaque-specific” antibodies recognizing theN-terminal Aβ(4-10) epitope from the serum Aβ-autoantibodies of anAlzheimer patient, isolated by Aβ(4-10)-epitope specific chromatography.IgG stands for immunoglobulin G. AD signifies Alzheimer's Disease.Affinity column: G5Aβ(4-10). kDa: Molecular weight in kilodalton.

FIG. 8: Molecular recognition mechanism of plaque-specific,plaque-disaggregating Aβ-antibodies recognizing the Aβ(4-10) epitope,and the anti-Aβ(21-37)-autoantibodies recognizing the Aβ(21-37)carboxyterminal epitope.

FIG. 9: Structure of Aβ(12-40) epitope-specific affinity column andexperimental procedure for isolation of anti-Aβ(21-37) autoantibodies.Cys-Aβ(12-40): Cysteine coupled Aβ(12-40) peptide.

FIG. 10: Analytical scheme and experimental procedures employed forsequence determination of affinity-isolated anti-Aβ(21-37)autoantibodies: N-terminal protein sequence analysis; 2D-electrophoreticseparation, in-gel proteolytic digestion and high resolution FTICR-MSidentification of constant region sequences; proteolytic digestion andHPLC separation of peptide fragments, followed by a) Edman sequencedeterminations; b) LC-MS/MS sequence determination; c) MALDI-TOFMSidentification of constant region partial sequences; MALDI-FTICR-MSidentification of constant/variable partial sequences.

FIG. 11: Analytical scheme of experimental procedure employed forassignment of heavy- and light chain sequence pairs of serum-IVIgGanti-Aβ(21-37) autoantibodies.

FIG. 12: 2-Dimensional SDS-gel electrophoretic separation of polyclonalanti-M(21-37) autoantibodies isolated from IVIgG; see FIG. 22 (a-c) foridentification and sequence determination of Aβ-antibody isoforms. DTT:Dithiothreitol. CHAPS:3-[(3-Cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate.

FIG. 13: 1D-SDS-PAGE isolation of heavy and light chains of serum IVIgGanti-Aβ(21-37) autoantibody. a) Reduction (10000×DTT); b) Alkylation (3×iodoacetamide/DTT). LMW: low molecular weight protein standard.

FIG. 14: 1D-Gel electrophoretic separation of HPLC-isolated heavy andlight chains of anti-Aβ(21-37) autoantibodies (serum-IVIgG) used forEdman sequence determinations. LMW: low molecular weight proteinstandard.

FIG. 15: 1D-Gel electrophoretic separation and blotting of serum IVIgGanti-Aβ(21-37) autoantibody heavy and light chains on PVDF membranes forEdman sequence determination.

FIG. 16: HPLC separation of heavy chain tryptic peptides. Isolatedpeptide fractions were subjected to a) Edman sequence analysis, b)LC-MS/MS sequence determination, c) direct MALDI-TOF-MS and d)MALDI-FTICR-MS analysis.

FIG. 17: HPLC separation of light chain tryptic peptides. Isolatedpeptide fractions were subjected to a) Edman sequence analysis, b)LC-MS/MS sequence determination, c) direct MALDI-TOF-MS and d) andMALDI-FTICR-MS analysis.

FIG. 18 (a-d): Edman sequence determination of HPLC-isolated heavy chaintryptic peptide, Serum_IVIG_G1_HC(1)_(—)1 c(348-359; EPQVYTLPPSR). 18 a:Standard. 18 b: Residue 1. 18 c: Residue 9. 18 d: Residue 11.

FIG. 19 (a-c): LC-MS/MS sequence determination of heavy chain trypticHPLC peptides, fraction 27 (a) and HPLC fraction 39, heavy chain CDR1peptide v(20-30). FIG. 19 a shows the total ion chromatogram, thepeptide fraction isolated at 1.3-2.1 min elution time is encircled inred. (b) ESI-mass spectrum of the peptide fraction isolated at 1.3-2.1min; (c) MS/MS fragment ion analysis of the doubly charged precursor ionselected, m/z 482.2.

FIG. 20 (a, b): MALDI-TOF-MS Identification of tryptic HPLC peptides. a)identification of tryptic peptide, fraction 50, heavy chain (138-151),mol. mass 1423; b) identification of peptide isolated in fraction 75,heavy chain (375-396), mol. mass 2544 Da.

FIG. 21 (a-c): MALDI-FTICR-Mass spectrometric identification of heavychain constant region tryptic peptides from HPLC fractions 47, 66, and96. (a) identification of 3 peptides isolated in fraction 47 denoted onthe molecular ion peaks, (349-359); (349-364), (137-151); (b)identification of 2 peptides in fraction 66, (260-278) and 279-292); (c)identification of peptide (306-321) in fraction 96.

FIG. 22 (a-c): MALDI-FT-ICR Identification of sequences comprising theserum IVIgG1 heavy chain constant regions, isolated from 2D-gel bandssubjected to in-gel tryptic digestion; spot 4 heavy chain (22 a), spot12 heavy chain (22 b) and spot 13 heavy chain (22 c) as illustrated inFIG. 12. Sequence determinations were performed using the NCBI database, at a mass accuracy threshold of 5-10 ppm.

FIG. 23 a to c: Table giving an overview about the identified andsequenced antibodies specific for a C-terminal part of Aβ-peptide, inparticular specific for Aβ(21-37). The table provides the name of theantibody chain sample, the source of the sample, the type ofimmunoglobulin chain sequenced, verified interactions of light and heavychains (indicated is the name of the partner chain as connection) aswell as confirmed isoforms of the respective immunoglobulin chain andthe type of CDR sequence identified for CDR1, CDR2 and CDR3. FIG. 23 a:IVIG_(1)_A′; IVIG_(2)_B′; IVIG_(3); IVIG_(4)_A; IVIG_(5)_B; IVIG_(6);IVIG_(7); IVIG_(8); Serum_(9); Serum_(10); Serum_(11); FIG. 23 b:IVIG_(12); IVIG_(13); IVIG_(14); IVIG_(15); IVIG_(16); IVIG_(17);IVIG_(18); IVIG_(19); IVIG_(20); IVIG_(21). FIG. 23 c: Serum_(22);Serum_(23); Serum_(24); Serum_(25).

FIG. 24 a to q: Table indicating the identified CDR types andcorresponding consensus sequences for all sequenced antibody chains. TheCDR numbers correspond with the numbering as used in FIG. 23 for CDRs.FIG. 24 a CDRs of heavy chains; FIG. 24 b: CDRs of light chains (lambdachain as well as kappa chain); FIG. 24 c: CDR consensus sequence for theCDR1 of the heavy chain, FIG. 24 d: CDR consensus sequence for the CDR2of the heavy chain, FIG. 24 e: CDR consensus sequence for the CDR3 ofthe heavy chain; FIG. 24 f: CDR consensus sequences for CDR1 of thekappa light chain, FIG. 24 g: CDR consensus sequences for CDR2 of thekappa light chain, FIG. 24 h: CDR consensus sequence for CDR3 of thekappa light chain, FIG. 24 i: preferred consensus sequence for CDR1 ofthe heavy chain, FIG. 24 j: more preferred consensus sequence for CDR1of the heavy chain, FIG. 24 k: more preferred consensus sequence forCDR1 of the heavy chain. FIG. 24 l: preferred consensus sequence forCDR2 of the heavy chain, FIG. 24 m: more preferred consensus sequencefor CDR2 of the heavy chain, FIG. 24 n: more preferred consensussequence for CDR2 of the heavy chain, FIG. 24 o: preferred consensussequence for CDR3 of the heavy chain, FIG. 24 p: more preferredconsensus sequence for CDR3 of the heavy chain, FIG. 24 q: morepreferred consensus sequence for CDR3 of the heavy chain.

FIGS. 25 a to l: Amino acid sequences of light chain variable regionsequences of anti-Aβ(21-37) autoantibodies from serum-IVIgG andindividual serum anti-Aβ(21-37) autoantibodies (see FIG. 23 for sequenceoverview). Annotation of codes for sequencing methods employed (Edman;Edman-protein N-terminal; MALDI-TOF-MS; MALDI-FTICR-MS, LC-MS/MS), andof CDR sequences is indicated on the bottom of each sequence. CDRs areindicated by boxes.

FIG. 25 a: amino acid sequence of light chain kappa variable region ofsample IVIG_(1)_A′ (SEQ ID NO:47).

FIG. 25 b: amino acid sequence of light chain kappa variable region ofsample IVIG_(2)_B′ (SEQ ID NO:48).

FIG. 25 c: amino acid sequence of light chain lambda variable region ofsample IVIG_(3) (SEQ ID NO:49).

FIG. 25 d: amino acid sequence of light chain kappa variable region ofsample IVIG_(6) (SEQ ID NO:50).

FIG. 25 e: amino acid sequence of light chain kappa variable region ofsample IVIG_(7) (SEQ ID NO:51).

FIG. 25 f: amino acid sequence of light chain kappa variable region ofsample IVIG_(8) (SEQ ID NO:52).

FIG. 25 g: amino acid sequence of light chain kappa variable region ofsample Serum_(9) (SEQ ID NO:53).

FIG. 25 h: amino acid sequence of light chain kappa variable region ofsample Serum_(10) (SEQ ID NO:54).

FIG. 25 i: amino acid sequence of light chain kappa variable region ofsample Serum_(11) (SEQ ID NO:55).

FIG. 25 j: amino acid sequence of light chain kappa variable region ofsample Serum_(9) (SEQ ID NO:145).

FIG. 25 k: amino acid sequence of light chain kappa variable region ofsample Serum_(9) (SEQ ID NO:146).

FIG. 25 l: amino acid sequence of light chain kappa variable region ofsample Serum_(9) (SEQ ID NO:147).

FIG. 26 a to q: Amino acid sequences of heavy chain variable regionsequences of anti-Aβ(21-37) autoantibodies from serum-IVIgG andindividual serum anti-Aβ(21-37) autoantibodies (see FIG. 23, sequenceoverview). Annotation of codes for sequencing methods employed (Edman;Edman-protein N-terminal; MALDI-TOF-MS; MALDI-FTICR-MS, LC-MS/MS), andof CDR sequences is indicated on the bottom of each sequence. CDRs areindicated by boxes.

FIG. 26 a: amino acid sequence of heavy chain variable region of sampleIVIG_(4)_A (SEQ ID NO:56).

FIG. 26 b: amino acid sequence of heavy chain variable region of sampleIVIG_(5)_B (SEQ ID NO:57).

FIG. 26 c: amino acid sequence of heavy chain variable region of sampleIVIG_(12) (SEQ ID NO:58).

FIG. 26 d: amino acid sequence of heavy chain variable region of sampleIVIG_(13) (SEQ ID NO:59).

FIG. 26 e: amino acid sequence of heavy chain variable region of sampleIVIG_(14) (SEQ ID NO:60).

FIG. 26 f: amino acid sequence of heavy chain variable region of sampleIVIG_(15) (SEQ ID NO:61).

FIG. 26 g: amino acid sequence of heavy chain variable region of sampleIVIG_(16) (SEQ ID NO:62).

FIG. 26 h: amino acid sequence of heavy chain variable region of sampleIVIG_(17) (SEQ ID NO:63).

FIG. 26 i: amino acid sequence of heavy chain variable region of sampleIVIG_(18) (SEQ ID NO:64).

FIG. 26 j: amino acid sequence of heavy chain variable region of sampleIVIG_(19) (SEQ ID NO:65).

FIG. 26 k: amino acid sequence of heavy chain variable region of sampleIVIG_(20) (SEQ ID NO:66).

FIG. 261: amino acid sequence of heavy chain variable region of sampleIVIG_(21) (SEQ ID NO:67).

FIG. 26 m: amino acid sequence of heavy chain variable region of sampleSerum_(22) (SEQ ID NO:68).

FIG. 26 n: amino acid sequence of heavy chain variable region of sampleSerum_(23) (SEQ ID NO:69).

FIG. 26 o: amino acid sequence of heavy chain variable region of sampleSerum_(24) (SEQ ID NO:70).

FIG. 26 p: amino acid sequence of heavy chain variable region of sampleSerum_(25) (SEQ ID NO:71).

FIG. 26 q: amino acid sequence of heavy chain variable region of sampleIVIG_(14) (SEQ ID NO:148).

FIG. 27 a to c: Amino acid sequences of the constant region of kappa andlambda light chains of anti-Aβ(21-37) autoantibody chains. The mutationof the LC-kappa-constant region sequence at V192L is indicated in boldletters.

FIG. 27 a: Complete amino acid sequence of constant region light chainkappa isoform 1 (SEQ ID NO:72).

FIG. 27 b: Complete amino acid sequence of constant region light chainkappa of isoform 2 (SEQ ID NO:73).

FIG. 27 c: Complete amino acid sequence of constant region light chainlambda (SEQ ID NO:74).

FIG. 28 a to c: Amino acid sequences and sequence isoforms of theconstant region identified for IVIgG-anti-Aβ(21-37) autoantibody heavychains. Amino acid mutations identified at F300Y, N301A (N-glycosylationsite), F304Y, G331A, D360E, L362M, S368T, and V401M are indicated bybold letters. The N-glycosylation consensus sequence and site at,N-301^(ST), are indicated by shaded box and bold grey letter.

FIG. 28 a: Complete amino acid sequence of constant region heavy chainof isoform 1 (SEQ ID NO:75).

FIG. 28 b: Complete amino acid sequence of constant region heavy chainof isoform 2 (SEQ ID NO:76).

FIG. 28 c: Complete amino acid sequence of constant region heavy chainisoform 3 (SEQ ID NO:77).

FIG. 29 a to p: Complete amino acid sequences of light chains ofanti-Aβ(21-37) autoantibodies from serum-IVIgG and individual serum (seeFIG. 23 for sample overview). Annotation of codes for sequencing methodsemployed (Edman; Edman-protein N-terminal; MALDI-TOF-MS; MALDI-FTICR-MS,LC-MS/MS), and of CDR sequences is indicated on the bottom of eachsequence. CDRs are indicated by boxes. Variable sequence domains, andsingle amino acid residues in the constant region sequences found withsingle site mutations are indicated in bold letters.

FIG. 29 a: complete amino acid sequence of light chain kappa of sampleIVIG_(1)_A′, constant region isoform 1 (SEQ ID NO:78).

FIG. 29 b: complete amino acid sequence of light chain kappa of sampleIVIG_(1)_A′, constant region isoform 2 (SEQ ID NO:79).

FIG. 29 c: complete amino acid sequence of light chain kappa of sampleIVIG_(2)_B′, constant region isoform 1 (SEQ ID NO:80).

FIG. 29 d: complete amino acid sequence of light chain kappa of sampleIVIG_(2)_B′, constant region isoform 2 (SEQ ID NO:81).

FIG. 29 e: complete amino acid sequence of light chain lambda of sampleIVIG_(3) (SEQ ID NO:82).

FIG. 29 f: complete amino acid sequence of light chain kappa of sampleIVIG_(6), constant region isoform 1 (SEQ ID NO:83).

FIG. 29 g: complete amino acid sequence of light chain kappa of sampleIVIG_(6), constant region isoform 2 (SEQ ID NO:84).

FIG. 29 h: complete amino acid sequence of light chain kappa of sampleIVIG_(7), constant region isoform 1 (SEQ ID NO:85).

FIG. 29 i: complete amino acid sequence of light chain kappa of sampleIVIG_(7), constant region isoform 2 (SEQ ID NO:86).

FIG. 29 j: complete amino acid sequence of light chain kappa of sampleIVIG_(8), constant region isoform 1 (SEQ ID NO:87).

FIG. 29 k: complete amino acid sequence of light chain kappa of sampleIVIG_(8), constant region isoform 2 (SEQ ID NO:88).

FIG. 291: complete amino acid sequence of light chain kappa of sampleSerum_(9), constant region isoform 1 (SEQ ID NO:89).

FIG. 29 m: complete amino acid sequence of light chain kappa of sampleSerum_(9), constant region isoform 2 (SEQ ID NO:90).

FIG. 29 n: complete amino acid sequence of light chain kappa of sampleSerum_(10), constant region isoform 1 (SEQ ID NO:91).

FIG. 29 o: complete amino acid sequence of light chain kappa of sampleSerum_(11), constant region isoform 1 (SEQ ID NO:92).

FIG. 29 p: complete amino acid sequence of light chain kappa of sampleSerum_(11), constant region isoform 2 (SEQ ID NO:93).

FIGS. 30-1 to 30-44: Complete amino acid sequences of heavy chains ofanti-Aβ(21-37) autoantibodies from serum-IVIgG and individual serum (seeFIG. 23 for sample overview). Annotation of codes for sequencing methodsemployed (Edman; Edman-protein N-terminal; MALDI-TOF-MS; MALDI-FTICR-MS,LC-MS/MS), and of CDR sequences is indicated on the bottom of eachsequence. CDRs are indicated by boxes. Variable sequence domains, andsingle amino acid residues in the constant region sequences found withsingle site mutations are indicated in bold letters. The N-glycosylationsite, N-301 is indicated in bold, grey letter.

FIG. 30-1: complete amino acid sequence of heavy chain of sampleIVIG_(4)_A, constant region isoform 1 (SEQ ID NO:94).

FIG. 30-2: complete amino acid sequence of heavy chain of sampleIVIG_(4)_A, constant region isoform 2 (SEQ ID NO:95).

FIG. 30-3: complete amino acid sequence of heavy chain of sampleIVIG_(4)_A, constant region isoform 3 (SEQ ID NO:96).

FIG. 30-4: complete amino acid sequence of heavy chain of sampleIVIG_(5)_B, constant region isoform 1 (SEQ ID NO:97).

FIG. 30-5: complete amino acid sequence of heavy chain of sampleIVIG_(5)_B, constant region isoform 2 (SEQ ID NO:98).

FIG. 30-6: complete amino acid sequence of heavy chain of sampleIVIG_(5)_B, constant region isoform 3 (SEQ ID NO:99).

FIG. 30-7: complete amino acid sequence of heavy chain of sampleIVIG_(12)_B, constant region isoform 1 (SEQ ID NO:100).

FIG. 30-8: complete amino acid sequence of heavy chain of sampleIVIG_(12)_B, constant region isoform 2 (SEQ ID NO:101).

FIG. 30-9: complete amino acid sequence of heavy chain of sampleIVIG_(12)_B, constant region isoform 3 (SEQ ID NO:102).

FIG. 30-10: complete amino acid sequence of heavy chain of sampleIVIG_(13), constant region isoform 1 (SEQ ID NO:103).

FIG. 30-11: complete amino acid sequence of heavy chain of sampleIVIG_(13), constant region isoform 2 (SEQ ID NO:104).

FIG. 30-12: complete amino acid sequence of heavy chain of sampleIVIG_(13), constant region isoform 3 (SEQ ID NO:105).

FIG. 30-13: complete amino acid sequence of heavy chain of sampleIVIG_(14), constant region isoform 1 (SEQ ID NO:106).

FIG. 30-14: complete amino acid sequence of heavy chain of sampleIVIG_(14), constant region isoform 2 (SEQ ID NO:107).

FIG. 30-15: complete amino acid sequence of heavy chain of sampleIVIG_(14), constant region isoform 3 (SEQ ID NO:108).

FIG. 30-16: complete amino acid sequence of heavy chain of sampleIVIG_(15), constant region isoform 1 (SEQ ID NO:109).

FIG. 30-17: complete amino acid sequence of heavy chain of sampleIVIG_(15), constant region isoform 2 (SEQ ID NO:110).

FIG. 30-18: complete amino acid sequence of heavy chain of sampleIVIG_(15), constant region isoform 3 (SEQ ID NO:111).

FIG. 30-19: complete amino acid sequence of heavy chain of sampleIVIG_(16), constant region isoform 1 (SEQ ID NO:112).

FIG. 30-20: complete amino acid sequence of heavy chain of sampleIVIG_(16), constant region isoform 2 (SEQ ID NO:113).

FIG. 30-21: complete amino acid sequence of heavy chain of sampleIVIG_(16), constant region isoform 3 (SEQ ID NO:114).

FIG. 30-22: complete amino acid sequence of heavy chain of sampleIVIG_(17), constant region isoform 1 (SEQ ID NO:115).

FIG. 30-23: complete amino acid sequence of heavy chain of sampleIVIG_(17), constant region isoform 2 (SEQ ID NO:116).

FIG. 30-24: complete amino acid sequence of heavy chain of sampleIVIG_(17), constant region isoform 3 (SEQ ID NO:117).

FIG. 30-25: complete amino acid sequence of heavy chain of sampleIVIG_(18), constant region isoform 1 (SEQ ID NO:118).

FIG. 30-26: complete amino acid sequence of heavy chain of sampleIVIG_(18), constant region isoform 2 (SEQ ID NO:119).

FIG. 30-27: complete amino acid sequence of heavy chain of sampleIVIG_(18), constant region isoform 3 (SEQ ID NO:120).

FIG. 30-28: complete amino acid sequence of heavy chain of sampleIVIG_(19), constant region isoform 1 (SEQ ID NO:121).

FIG. 30-29: complete amino acid sequence of heavy chain of sampleIVIG_(19), constant region isoform 2 (SEQ ID NO:122).

FIG. 30-30: complete amino acid sequence of heavy chain of sampleIVIG_(19), constant region isoform 3 (SEQ ID NO:123).

FIG. 30-31: complete amino acid sequence of heavy chain of sampleIVIG_(20), constant region isoform 1 (SEQ ID NO:124).

FIG. 30-32: complete amino acid sequence of heavy chain of sampleIVIG_(20), constant region isoform 2 (SEQ ID NO:125).

FIG. 30-33: complete amino acid sequence of heavy chain of sampleIVIG_(20), constant region isoform 3 (SEQ ID NO:126).

FIG. 30-34: complete amino acid sequence of heavy chain of sampleIVIG_(21), constant region isoform 1 (SEQ ID NO:127).

FIG. 30-35: complete amino acid sequence of heavy chain of sampleIVIG_(21), constant region isoform 2 (SEQ ID NO:128).

FIG. 30-36: complete amino acid sequence of heavy chain of sampleIVIG_(21), constant region isoform 3 (SEQ ID NO:129).

FIG. 30-37: complete amino acid sequence of heavy chain of sampleSerum_(22), constant region isoform 1 (SEQ ID NO:130).

FIG. 30-38: complete amino acid sequence of heavy chain of sampleSerum_(22), constant region isoform 2 (SEQ ID NO:131).

FIG. 30-39: complete amino acid sequence of heavy chain of sampleSerum_(23), constant region isoform 1 (SEQ ID NO:132).

FIG. 30-40: complete amino acid sequence of heavy chain of sampleSerum_(23), constant region isoform 2 (SEQ ID NO:133).

FIG. 30-41 complete amino acid sequence of heavy chain of sampleSerum_(24), constant region isoform 1 (SEQ ID NO:134).

FIG. 30-42: complete amino acid sequence of heavy chain of sampleSerum_(24), constant region isoform 3 (SEQ ID NO:135).

FIG. 30-43 complete amino acid sequence of heavy chain of sampleSerum_(24), constant region isoform 1 (SEQ ID NO:136).

FIG. 30-44: complete amino acid sequence of heavy chain of sampleSerum_(24), constant region isoform 3 (SEQ ID NO:137).

FIG. 31: Table illustrating the conserved nature of the N-terminus ofkappa light chain of the antibodies sequenced for the present invention.Indicated are the 6 types of N-terminal sequences, consisting of 18amino acid residues, which were identified in the kappa light chainsequences of the antibodies of the present invention.

FIGS. 32 a and b: Scheme of intra- and inter-disulfide linkages ofanti-Aβ(21-37) autoantibodies for HC-LC-kappa and HC-LC-lambdaconnections. HC intradisulfide linkages are C21-C96, C148-C204,C265-C325, C371-C429; LC-kappa-intradisulfide linkages are C23-C89,C135-C195; LC-lambda-intradisulfide linkages are C22-C92, C142-C201;HC-HC interdisulfide linkages are C230-C230, C233-C33; HC-LC-kappa- andHC-LC-lambda-interdisulfide linkages are C224-C215 and C224-C219,respectively. 32 a: IVIgG_LC(1)_HC(1); 32 b: IVIG_HC(1)_LCλ(3).

FIG. 33: Western blot showing that the recombinant anti-Aβ(21-37)autoantibody CSL-Clone 7 immunoprecipitates oligomeric forms of Aβ1-40as described in Example 6. The antibody Bam 90.1 (Sigma Aldrich Cat#A8978 binding to Aβ(13-28)) was used to detect the immunoprecipitatedAβ.

FIG. 34 a: Molecular confirmation of epitope recognition specificity ofAβ-autoantibody. Illustrated is the affinity of 3 syntheticAβ-polypeptides Aβ(4-10), Aβ(20-30) and Aβ(20-37) towardsanti-Aβ-autoantibodies isolated from serum of healthy (non-AD controlindividuals) donors, A and B, by MALDI-mass spectrometry.Affinity-purified antibodies were immobilized on NHS-sepharose asdescribed in Example 2A (Aβ12-40). Equimolar mixtures (5 μmol mixturesof synthetic Aβ-peptides in aqueous PBS buffer solution, pH 7) werebound to the antibodies after mass spectrometric analysis (MALDI-MS ofpeptide mixture, upper panel). MALDI-MS of the supernatant washingfraction revealed the N-terminal Aβ(4-10) epitope signal as thepredominant ion (confirming the lack of binding of N-terminal Aβ; middlepanel), and washing was continued until no MS signal was detectable.After elution with 0.1% trifluoroacetic acid, the Aβ(20-37) peptide wasidentified as the only polypeptide capable of binding to theautoantibodies (lower panel). All MS determinations were made with aBroker Bilflex MALDI-TOF spectrometer.

FIG. 34 b: Mass spectrograms showing epitope specificity ofAβ-autoantibody. Immobilized Aβ(21-37) autoantibodies purified fromIVIgG according to Example 2A were incubated with a synthetic Aβ(12-40)polypeptide. The elution profiles were analyzed via MS as above. Thedata show that the Aβ(21-37) autoantibodies specifically bound theAβ(12-40) polypeptide.

FIG. 34 c: Mass spectrograms showing epitope specificity ofAβ-autoantibody. Immobilized Aβ(21-37) autoantibodies purified fromIVIgG were incubated with synthetic Aβ-polypeptides Aβ(25-35), Aβ(17-28)and Aβ(31-40). The data show that the Aβ(21-37) autoantibodies boundnone of the Aβ partial polypeptides.

FIG. 34 _(—) d to 34 _(—) l: Mass spectrograms showing epitopespecificity of Aβ-autoantibody. Immobilized Aβ(21-37) autoantibodiespurified from IVIgG and immobilized antibody ACA (see example 5) wereincubated with synthetic polypeptides Aβ(4-10), Aβ(17-28), Aβ(12-40) andAβ(20-37). The data show that both the immobilized ACA antibody and theimmobilized Aβ(21-37) autoantibodies bind to Aβ(1-40) and to Aβ(12-40)but that only the immobilized Aβ(21-37) autoantibodies specifically bindto Aβ(20-37). Both immobilized antibodies did not bind Aβ(17-28).Specifically, FIG. 34 _(—) d shows that mab ACA does not bind toAβ(4-10), FIG. 34 _(—) e shows that mab ACA does not bind to Aβ(17-28),FIG. 34 _(—) f shows that mab ACA does bind to Aβ(12-40), FIG. 34 _(—) gshows that mab ACA does not bind to Aβ(20-37), FIG. 34 _(—) h shows thatmab ACA does bind to Aβ(1-40), FIG. 34 _(—) i shows that Aβ(21-37)autoantibodies do bind to Aβ(1-40), FIG. 34 j shows that Aβ(21-37)autoantibodies do bind to Aβ(12-40), FIG. 34 _(—) k shows that Aβ(21-37)autoantibodies do not bind to Aβ(17-28) and FIG. 34 _(—) l shows thatAβ(21-37) autoantibodies do bind to Aβ(20-37).

FIG. 35: Serum ELISA for determination of anti-Aβ(21-37) autoantibodies.BSA is bovine serum albumin. HRP is horseradish peroxidase. OPD iso-phenylenediamine. IgG stands for immunoglobulin G.

FIG. 36: ELISA determination of Aβ-autoantibody (from IVIgG). IVIgGstands for intravenous IgG preparation. The ELISA was carried out withAβ(1-40) coated on 96-well plate, and dilutions of Aβ-antibody wereadded, and determined with anti-human horseradish peroxidase-conjugatedsecondary antibody. Aβ-antibody quantifications were performed with a 1μg/μl stock solution, using a BSA reference curve for calibration. Thepercentage indicated represents the Aβ-antibody concentrations in IVIgGfrom two separate ELISA determinations.

FIG. 37: Western blot showing that affinity purified IVIgG according toExample 4 immunoprecipitates oligomeric forms of Aβ1-40 as described inExample 6. The antibody Bam 90.1 (Sigma Aldrich Cat# A8978 binding toAβ(13-28)) was used to detect the immunoprecipitated Aβ.

FIG. 38: Bar graph representing the mean total plaque area per antibodyused in an AD animal model as described in Example 13. Black columnsrepresent the plaque area in the cortex, white bars represent the plaquearea in the hippocampus. Plaque area was measured using the Nikon NISElements Software on pictures of immunostained brain slices of thetreated animals. The measured plaque area of the CSL 360- or CSL Clone7-treated animals (N=2) were averaged for both animals for comparisonwith the affinity-purified IVIgG-treated animal (N=1).

FIG. 39: ELISA data showing that anti-Aβ(21-37) autoantibody CSL-Clone 7binds to Aβ(1-40) and to Aβ(12-40) peptides but not to Aβ(4-10) asdiscussed in Example 9D.

FIG. 40: The effect of 3 different Aβ-specific antibodies: Aβ affinitycolumn purified human IVIgG (as described in Example 4), the humanmonoclonal Aβ autoantibody CSL Clone 7 (as described in Example 5) andhumanized murine monoclonal antibody raised against a midterminal Aβpeptide sequence (AK ACA, as described in Example 5) to inhibit Aβfibril formation as measured by THT fluorescence staining as describedin Example 10. The fluorescence of the THT assay is proportional tofibrillar Ab and was used to assess fibril morphology. The fluorescenceof Aβ(1-40) incubated in the presence of a nonspecific human monoclonal(CSL360) was set to 100%.

FIG. 41: Dot Blot Analysis as described in Example 11A. Samples weretested with control antibodies (6E10, Bam90.1, CSL Clone 7, affinitypurified IVIG, ACA), serum from an AD-patient (AD1), serum from an agematched healthy human individual (K4) as described in Example 11A

FIG. 42: IgG from serum samples (one AD positive sample and oneage-matched control sample) after purification on Protein G (Pierce)were loaded on an Aβ(1-16) column, washed and eluted with 100 mM GlycinepH 2.8. The eluate was analyzed in a Biotin-G₅-Aβ(4-10) ELISA asdescribed in Example 11B.

FIG. 43: Binding to Aβ(1-40 Cys) dimer as opposed to Aβ(1-40) monomer asdescribed in Example 12C for the recombinant Aβ(21-37) autoantibodies55/61, 146/61 and the control antibody ACA

FIG. 44: Binding to Aβ(1-40 Cys) dimer as opposed to Aβ(1-40) monomer asdescribed in Example 12C for the recombinant Aβ(21-37) autoantibodies54/61, 47/56, 51/60 and 53/60.

FIG. 45: Binding to Aβ(1-40 Cys) dimer as opposed to Aβ(1-40) monomer asdescribed in Example 12C for the recombinant Aβ(21-37) autoantibodies146/60, 52/60, 53/148 and 145/60.

FIG. 46: Tricine Gel protein blot analysis of β amyloid peptides.Protein visualisation of β amyloid peptide was done using standardcoomassie staining techniques as described in the Novex gel manual(Invitrogen) and deep purple reagent for high sensitivity. Deep purple(GE, Sweden) was visualised using a Typhoon scanner as per manufacturerinstruction.

FIG. 47: Western blot comparing the binding of the antibodies 6E10, ACAand CSL Clone 7 to Aβ(1-40) monomer, Aβ(1-40) oligomer and Aβ(1-40 Cys)oligomer as described in Example 12D.

FIG. 48 a: Immunohistochemistry of a human brain sample of a patientsuffering from Alzheimers disease using the 6F3D anti β-amyloid antibody(Dako) as primary antibody, and the Vectastain® M.O.M.-Kit (HRP antimouse) as a detection system. A specific immunostaining is detectable inthe vessel wall (arrow) as well as the Alzheimer-plaque (arrowheads).This immunostaining serves as positive control.

FIG. 48 b: Immunohistochemistry of a human brain sample of a patientsuffering from Alzheimers disease using the ACA antibody as primaryantibody, and the Vectastain® Elite ABC Kit (HRP anti human) asdetection system. A specific immunostaining is detectable in the vesselwall (arrow).

FIG. 48 c: Immunohistochemistry of a human brain sample of a patientsuffering from Alzheimers disease using the ACA antibody as primaryantibody, and the Vectastain® Elite ABC Kit (HRP anti human) asdetection system. A specific immunostaining is detectable in the vesselwall (arrow) as well as the Alzheimer-plaque (arrowheads, insert).

FIG. 48 d: Immunohistochemistry of a human brain sample of a patientsuffering from Alzheimers disease and CAA using the affinity purifiedIVIgG as primary antibody, and the Vectastain® Elite ABC Kit (HRP antihuman) as detection system. No specific immunostaining is detectable inthe vessel wall (arrow).

FIG. 48 e: Immunohistochemistry of a human brain sample of a patientsuffering from Alzheimers disease using the clone 7 antibody as primaryantibody, and the Vectastain® Elite ABC Kit (HRP anti human) asdetection system. No specific immunostaining is detectable in the vesselwall (arrow).

FIG. 48 f: Immunohistochemistry of a human brain sample of a patientsuffering from Alzheimers disease using the CSL 360 antibody as primaryantibody, and the Vectastain®Elite ABC Kit (HRP anti human) as detectionsystem. No specific immunostaining is detectable in the vessel wall(arrow), but the blood in the vessel lumen show unspecific backgroundstaining.

FIG. 49: Toxicity of Aβ-oligomers for human neuroblastoma cells(SH-Sy5y) was tested as described in Example 4. The experiment wasrepeated with affinity purified IVIgG (purified as described above, mabCSL Clone 7 (see Example 5). As a negative control the antibodies CSL360(see Example 10) or no antibody was used. A positive control antibodyused was ACA (see Example 10). Results clearly show a dose dependenteffectiveness of protecting cells from the neurotoxic effects of Aβoligomers of both the affinity purified mab CSL Clone 7 and affinitypurified IVIgG.

DETAILED DESCRIPTION

Aβ-autoantibodies were isolated from the serum of AD patients andhealthy controls or pooled commercially obtainable serum immunoglobulin(IVIgG). The cDNA and amino acid sequences of the variable regions ofthe heavy and light chains were determined and all possible pairings ofheavy and light chains were expressed in mammalian cells. A number ofthese pairings were found to bind with higher affinity to Aβ dimers thanto Aβ monomers and one of these, CSL clone 7, was shown to possessbiological activities potentially useful for the treatment of AD. Theinventors further discovered that the CDRs of anti-Aβ(21-37)autoantibodies are highly homologous. Accordingly, consensus CDRs weredetermined and used to prepare human anti-β-amyloid antibodies usefulfor preventing or treating neurodementing diseases like AD. Theinventors also surprisingly discovered that AD patients, as compared tohealthy controls, have an increased antibody titer against Aβ(4-10) anda decreased antibody titer against Aβ(21-37). Thus, the inventorsdiscovered not only means for detecting and measuring the progression ofa neurodementing disease like AD, but also methods for delaying theonset or progression of AD. Kits for detecting and measuring theprogression of neurodementing diseases, like AD, also are provided.

DEFINITIONS

The term “Aβ polypeptide” as used herein, defines a polypeptide havingthe amino acid sequence SEQ ID NO:01 or fragments thereof. Suchfragments in particular comprise polypeptides having the sequence SEQ IDNO:02.

The term “antibody”, as used herein, comprises also derivatives and/orfragments of antibodies. Such derivatives or fragments of antibodiesretain the antigen binding properties of the intact antibody, but whichlack some sequences of the intact antibody, for example the Fc-domain.Examples for such derivatives or fragments include, but are not limitedto, Fab or F(ab′)₂ fragments, which are obtainable via enzymatic digestof antibodies with papain or pepsin protease, respectively, single chainvariable fragments (scFv), Fv fragments, minibodies and diabodies.

The term “autoantibody” or “autoantibodies”, as used herein, refers ingeneral to antibodies which are directed against epitopes on proteins ofthe human body and which can be found in the blood or cerebrospinalfluid of a human subject without prior immunization with the respectiveantigen. Meanwhile, the term “anti-Aβ(21-37) autoantibody” refers toautoantibodies that bind to an Aβ peptide comprising Aβ(21-37) (SEQ IDNO:2) and shield said SEQ ID NO 2 from proteolytic digestion. Suchanti-Aβ(21-37) autoantibodies also bind with a higher affinity to dimersof Aβ than to corresponding monomers of Aβ.

The term “CDR”, as used herein, refers to Complementarity DeterminingRegions. Usually 3 of such CDR-regions (CDR1, CDR2, CDR3) can be foundin the variable region on the light chain as well as on the heavy chainof an antibody. Each of these six hypervariable regions can contributeto the antigen specificity of the antibody. However, as used herein, theterm CDR does not imply that the molecule referred to is in fact anantibody. Rather, the term is considered to designate a sequencecontributing to the specific binding of a polypeptide according to theinvention to a C-terminal part of full length Aβ polypeptide (Aβ1-40),in particular contributing to the binding to Aβ(21-37) polypeptide.Consequently, also derivatives of antibodies or other polypeptidesengineered for binding to said epitope can exhibit CDRs.

The term “consensus CDR” as used herein refers to a single sequencederived by aligning two or more sequences for a given CDR according tothe Kabat numbering system. (see Kabat, E. A., Wu, T. T., Perry, H. M.,Gottesman, K. S. & Foeller, C. (1991) Sequences of Proteins ofImmunological Interest (Department of Health and Human Services, PublicHealth Service, National Institutes of Health, Bethesda, Md.) NH Publ.No 91-3442 5th Ed. and R. Kontermann, S. Dübel (eds.), AntibodyEngineering; Springer Lab Manual Series; Springer, Heidelberg 2001, bothof which are hereby incorporated by reference.) Accordingly, for eachamino acid position of the “consensus CDR”, the identity of amino acidswhich can occur at that position is determined. CDR designations as wellas amino acid insertions are made according to Kabat numbering.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161.

The term “epitope” or “epitope peptide”, as used herein, generallyrefers to a polypeptide comprising the molecular recognition peptidesequence or structure derived from an antigen, that is bound by aspecific antibody. It is an immunological determinant group of anantigen which is specifically recognized by the antibody. An epitope maycomprise at least 5, preferably at least 8 amino acids in a spatial ordiscontinuous conformation. An epitope may also comprise a singlesegment of a polypeptide chain comprising a continuous linear amino acidsequence with a minimal length of approx. 5 amino acids.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the VH VL dimer. Collectively, the six CDRs confer antigenbinding specificity to the antibody.

The term “moiety”, as used herein, refers to a portion of a polypeptidewith distinct function(s). Such moieties can provide for a structural orfunctional feature which is normally not present in the rest of thepolypeptide or which feature is enhanced by this moiety. Such functionalor structural moieties can for example provide binding, stabilization ordetection of the polypeptide. The moiety can be a polypeptide on its ownor can be any other compound, which provides the desired function(s) tothe polypeptide. Said moiety is stably associated with the polypeptide,in particular covalently coupled to the polypeptide. The term moiety, asused herein, does not confer any information about the size of thisportion in comparison to the polypeptide itself. The moiety can besmaller, equally sized or larger than the polypeptide it is coupled to.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a specific antigenic site or epitope, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different epitopes.

The terms “neurodementing diseases”, “dementing disorders” or“neurodementia diseases of AD-type”, as used herein, refer to diseasesselected from Alzheimer's disease, Down's syndrome, Dementia with Lewybodies, fronto-temporal dementia as well as to disorders such ascerebral amyloid angiopathy and amyloidoses.

The term “oligomerized”, “oligomers” and “oligomeric” refers tomultimers of Aβ comprising also Aβ dimers, trimers, tetramers and higheroligomers but not to Aβ fibrils.

The term “plaque-specific” antibody, as used herein, refers to anantibody directed against the Aβ(4-10) epitope of Aβ polypeptide.

The term “polypeptide”, as used herein, refers to a polypeptide chain ofat least 5 amino acid residues. The term also refers to an assembly ofmore than one polypeptide chain, e.g. an assembly of four polypeptidechains, such as an IgG antibody.

The term “paratope” or “paratope peptide”, as used herein, generallydefines a molecular recognition peptide sequence derived from a specificmonoclonal or polyclonal antibody. This recognition peptide sequenceexerts specific binding properties to an antigen epitope, and maycomprise variable and/or constant region partial sequences of anantibody.

“Specifically binding to epitope X” as used herein, refers to theproperty of an antibody to bind to a particular epitope, e.g. epitope X,with a higher affinity than to other epitopes.

“Specifically binding to oligomeric structures of Aβ”, as used herein,means that the respective antibody binds to oligomeric structures of Aβwith a higher affinity than to monomeric structures of Aβ.

A “therapeutically effective” amount of Aβ antibody refers to the dosagethat provides the specific pharmacological response for which theantibody is administered in a significant number of subjects in need ofsuch treatment. A therapeutically effective amount can be determined byprevention or amelioration of adverse conditions or symptoms of theneurodementing disease being treated. The appropriate dosage will varydepending, for example, on the type, stage, and severity of the disease,as well as on the mode of administration. It is emphasized that a“therapeutically effective amount” administered to a particular subjectin a particular instance may not be effective for 100% of patientstreated for a specific disease, even though such dosage is deemed a“therapeutically effective amount” by skilled practitioners.

The term “titer” denotes a measurement of the amount or concentration ofa particular antibody in a sample, typically blood.

The terms “treatment,” “treating,” “treat,” and the like refer toobtaining a desired pharmacological and/or physiologic effect. Theeffect can be prophylactic in tennis of completely or partiallypreventing a disease or symptom thereof and/or can be therapeutic interms of a partial or complete stabilization or cure for a diseaseand/or adverse effect attributable to the disease. “Treatment” coversany treatment of a disease in a mammal, particularly a human, andincludes: (a) preventing the disease or symptom from occurring in asubject which can be predisposed to the disease or symptom but has notyet been diagnosed as having it; (b) inhibiting the disease symptom,i.e., arresting its development; or (c) relieving the disease symptom,i.e., causing regression of the disease or symptom.

Antibodies

In one aspect the present invention relates to a polypeptide bindingspecifically to the epitope Aβ(21-37) (SEQ ID NO 2).

A polypeptide according to the present invention can be for example anantibody, an antibody fragment, or any other polypeptide, binding to aC-terminal part of full length AD polypeptide (Aβ1-40), in particular tothe epitope denoted in Aβ(21-37) (SEQ ID NO:2).

In one aspect, the inventive polypeptides are capable of bindingspecifically to a polypeptide comprising the epitope Aβ(21-37) (SEQ IDNO 2) of amyloid beta, in particular under physiological conditions,e.g. pH about 7.4, salt concentrations about 50 to about 150 mM in PBS.In one embodiment, the polypeptides have a relative dissociationconstant, under in vitro conditions, (relative KD; reflecting in vitroresults but not necessarily identical values under in vivo conditions)of at least about 10-5 M, about 10-6 M, about 10-7 M, about 10-8, about10-9 M, about 10-10M, about 10-11 M, about 10-12 M, or higher. Forexample, the relative dissociation constant of the binding of apolypeptide to the epitope denoted in Aβ(21-37) (SEQ ID NO:2) can bebetween about 10-8 M to about 10-12 M, in particular around 1 to 50×10-9M. Such dissociation rate constants can be determined readily usingkinetic analysis techniques such as surface plasmon resonance (BIAcoreor Biosensor), using general procedures outlined by the manufacturer orother methods known in the art.

In another aspect, it was surprisingly discovered that polypeptidesspecific for Aβ(21-37) are highly homologous. Thus, polypeptides areprovided that comprise sequences selected from consensus CDR sequences.Accordingly, in one embodiment, a polypeptide according to the presentinvention can comprise a CDR sequence having a sequence as denoted inany of the consensus sequences SEQ ID NOs: 6 to 8.

SEQ ID NOs: 6 to 8 represent consensus sequences for CDR regions of theheavy chain of an antibody having the ability to bind to SEQ ID NO:2.The consensus sequences are derived from the sequence informationderived from the antibodies, which have been identified by the inventorsto bind specifically to SEQ ID NO:2. In particular, the identifiedconsensus sequences for the CDR regions of both the heavy chain (i.e.SEQ ID NOs: 6 to 8 and SEQ ID Nos 153 to 161) and the light chain (i.e.SEQ ID NOs: 9 to 11) were derived from naturally-occurring, humanantibodies isolated as described in Example 2A.

SEQ ID NO:6 represents a CDR1 consensus sequence for the heavy chain,wherein

the amino acid at position 1 of SEQ ID NO:6 which is at the Kabatposition H31 can be Ser, Gly or Asn,the amino acid at position 2 of SEQ ID NO:6 which is the amino acid atthe Kabat position 8 H32 is Tyr,the amino acid at position 3 of SEQ ID NO:6 which is the amino acid atthe Kabat position H33 can be Trp or Asp,the amino acid at position 4 of SEQ ID NO:6 which is the amino acid atthe Kabat position H34 is Met andthe amino acid at position 5 of SEQ ID NO:6 which is the amino acid atthe Kabat position H35 can be Ser or His.

SEQ ID NO:153 represents a preferred CDR1 consensus sequence for theheavy chain, wherein

the amino acid at position 1 of SEQ ID 153 which is at the Kabatposition H31 can be Asn or Ser,the amino acid at position 2 of SEQ ID 153 which is the amino acid atthe Kabat position 8 H32 is Tyr,the amino acid at position 3 of SEQ ID NO: 153 which is the amino acidat the Kabat position H33 can be Asp or Trp,the amino acid at position 4 of SEQ ID NO: 153 which is the amino acidat the Kabat position H34 is Met andthe amino acid at position 5 of SEQ ID NO: 153 which is the amino acidat the Kabat position H35 can be His or Ser.

SEQ ID NO:154 represents a more preferred CDR1 consensus sequence forthe heavy chain, wherein

the amino acid at position 1 of SEQ ID NO: 154 which is at the Kabatposition H31 of SEQ ID NO:6 is Asn,the amino acid at position 2 of SEQ ID NO: 154 which is the amino acidat the Kabat position 8 H32 is Tyr,the amino acid at position 3 of SEQ ID NO: 154 which is the amino acidat the Kabat position H33 is Asp,the amino acid at position 4 of SEQ ID NO: 154 which is the amino acidat the Kabat position H34 is Met andthe amino acid at position 5 of SEQ ID NO: 154 which is the amino acidat the Kabat position H35 is His.

SEQ ID NO:155 represents a more preferred CDR1 consensus sequence forthe heavy chain, wherein

the amino acid at position 1 of SEQ ID NO: 155 which is at the Kabatposition H31 is Ser,the amino acid at position 2 of SEQ ID NO: 155 which is the amino acidat the Kabat position 8 H32 is Tyr,the amino acid at position 3 of SEQ ID NO: 155 which is the amino acidat the Kabat position H33 can be Trp or Asp,the amino acid at position 4 of SEQ ID NO: 155 which is the amino acidat the Kabat position H34 is Met andthe amino acid at position 5 of SEQ ID NO: 155 which is the amino acidat the Kabat position H35 is Ser.

SEQ ID NO:7 represents a CDR2 consensus sequence for the heavy chain,wherein

the amino acid at position 1 of SEQ ID NO:7 which is the amino acid atthe Kabat position H50 can be Ser or Arg or Glu,the amino acid at position 2 of SEQ ID NO:7 which is the amino acid atthe Kabat position H51 can be Val or Ile,the amino acid at position 3 of SEQ ID NO:7 which is the amino acid atthe Kabat position H52 can be Lys or Gly or Asn,the amino acid at position 4 of SEQ ID NO:7 which is the amino acid atthe Kabat position H52a can be Gln or no amino acid,the amino acid at position 5 of SEQ ID NO:7 which is the amino acid atthe Kabat position H53 can be Asp or Phe or Thr or Arg,the amino acid at position 6 of SEQ ID NO:7 which is the amino acid atthe Kabat position H54 can be Gly or Phe or Ala or Ser,the amino acid at position 7 of SEQ ID NO:7 which is the amino acid atthe Kabat position H55 can be Ser or Gly,the amino acid at position 8 of SEQ ID NO:7 which is the amino acid atthe Kabat position H56 can be Glu or Gly or Arg or Asp or Ala,the amino acid at position 9 of SEQ ID NO:7 which is the amino acid atthe Kabat position H57 can be Lys or Pro or Ser or Thr or Arg,the amino acid at position 10 of SEQ ID NO:7 which is the amino acid atthe Kabat position H58 can be Tyr or Leu or Ala or Asn,the amino acid at position 11 of SEQ ID NO:7 which is the amino acid atthe Kabat position H59 can be Tyr or Ala,the amino acid at position 12 of SEQ ID NO:7 which is the amino acid atthe Kabat position H60 can be Val or Thr or Ala or Asn,the amino acid at position 13 of SEQ ID NO:7 which is the amino acid atthe Kabat position H61 can be Asp or Gly or Pro,the amino acid at position 14 of SEQ ID NO:7 which is the amino acid atthe Kabat position H62 is Ser,the amino acid at position 15 of SEQ ID NO:7 which is the amino acid atthe Kabat position H63 can be Val or Leu,the amino acid at position 16 of SEQ ID NO:7 which is the amino acid atthe Kabat position H64 is Lys andthe amino acid at position 17 of SEQ ID NO:7 which is the amino acid atthe Kabat position H65 can be Gly or Ser.

SEQ ID NO:156 represents a preferred CDR2 consensus sequence for theheavy chain, wherein

the amino acid at position 1 of SEQ ID NO:156 which is the amino acid atthe Kabat position H50 can be Arg or Ser or Glu,the amino acid at position 2 of SEQ ID NO:156 which is the amino acid atthe Kabat position H51 can be Ile or Val,the amino acid at position 3 of SEQ ID NO:156 which is the amino acid atthe Kabat position H52 can be Gly or Lys or Asn,the amino acid at position 4 of SEQ ID NO:156 which is the amino acid atthe Kabat position H52a can be Gln or no amino acid,the amino acid at position 5 of SEQ ID NO:156 which is the amino acid atthe Kabat position H53 can be Thr or Asp or Arg,the amino acid at position 6 of SEQ ID NO:156 which is the amino acid atthe Kabat position H54 can be or Ala or Gly or Ser,the amino acid at position 7 of SEQ ID NO:156 which is the amino acid atthe Kabat position H55 can be Gly or Ser,the amino acid at position 8 of SEQ ID NO:156 which is the amino acid atthe Kabat position H56 can be Arg or Asp or Glu or Ala,the amino acid at position 9 of SEQ ID NO:156 which is the amino acid atthe Kabat position H57 can be Thr or Arg or Lys,the amino acid at position 10 of SEQ ID NO:156 which is the amino acidat the Kabat position H58 can be Asn or Tyr,the amino acid at position 11 of SEQ ID NO:156 which is the amino acidat the Kabat position H59 is Tyr,the amino acid at position 12 of SEQ ID NO:156 which is the amino acidat the Kabat position H60 can be Asn, Ala or Val,the amino acid at position 13 of SEQ ID NO:156 which is the amino acidat the Kabat position H61 can be Pro or Gly or Asp,the amino acid at position 14 of SEQ ID NO:156 which is the amino acidat the Kabat position H62 is Ser,the amino acid at position 15 of SEQ ID NO:156 which is the amino acidat the Kabat position H63 can be Leu or Val,the amino acid at position 16 of SEQ ID NO:156 which is the amino acidat the Kabat position H64 is Lys andthe amino acid at position 17 of SEQ ID NO:156 which is the amino acidat the Kabat position H65 can be Gly or Ser.

SEQ ID NO:157 represents a more preferred CDR2 consensus sequence forthe heavy chain, wherein

the amino acid at position 1 of SEQ ID NO:157 which is the amino acid atthe Kabat position H50 can be Arg or Glu,the amino acid at position 2 of SEQ ID NO:157 which is the amino acid atthe Kabat position H51 is Ile,the amino acid at position 3 of SEQ ID NO:157 which is the amino acid atthe Kabat position H52 can be Gly or Asn,the amino acid at position 4 of SEQ ID NO:157 which is the amino acid atthe Kabat position H53 can be Thr or Arg,the amino acid at position 5 of SEQ ID NO:157 which is the amino acid atthe Kabat position H54 can be Ala or Ser,the amino acid at position 6 of SEQ ID NO:157 which is the amino acid atthe Kabat position H55 is Gly,the amino acid at position 7 of SEQ ID NO:157 which is the amino acid atthe Kabat position H56 can be Arg or Asp or Ala,the amino acid at position 8 of SEQ ID NO:157 which is the amino acid atthe Kabat position H57 can be Thr or Arg,the amino acid at position 9 of SEQ ID NO:157 which is the amino acid atthe Kabat position H58 can be Asn or Tyr,the amino acid at position 10 of SEQ ID NO:157 which is the amino acidat the Kabat position H59 is Tyr,the amino acid at position 11 of SEQ ID NO:157 which is the amino acidat the Kabat position H60 can be Asn or Ala,the amino acid at position 12 of SEQ ID NO:157 which is the amino acidat the Kabat position H61 can be Pro or Gly,the amino acid at position 13 of SEQ ID NO:157 which is the amino acidat the Kabat position H62 is Ser,the amino acid at position 14 of SEQ ID NO:157 which is the amino acidat the Kabat position H63 can be Leu or Val,the amino acid at position 15 of SEQ ID NO:157 which is the amino acidat the Kabat position H64 is Lys andthe amino acid at position 16 of SEQ ID NO:157 which is the amino acidat the Kabat position H65 can be Gly or Ser.

SEQ ID NO:158 represents a more preferred CDR2 consensus sequence forthe heavy chain, wherein

the amino acid at position 1 of SEQ ID NO:158 which is the amino acid atthe Kabat position H50 is Ser,the amino acid at position 2 of SEQ ID NO:158 which is the amino acid atthe Kabat position H51 is Val,the amino acid at position 3 of SEQ ID NO:158 which is the amino acid atthe Kabat position H52 is Lys,the amino acid at position 4 of SEQ ID NO:158 which is the amino acid atthe Kabat position H52a is Gln,the amino acid at position 5 of SEQ ID NO:158 which is the amino acid atthe Kabat position H53 is Asp,the amino acid at position 6 of SEQ ID NO:158 which is the amino acid atthe Kabat position H54 is Gly,the amino acid at position 7 of SEQ ID NO:158 which is the amino acid atthe Kabat position H55 is Ser,the amino acid at position 8 of SEQ ID NO:158 which is the amino acid atthe Kabat position H56 is Glu,the amino acid at position 9 of SEQ ID NO:158 which is the amino acid atthe Kabat position H57 is Lys,the amino acid at position 10 of SEQ ID NO:158 which is the amino acidat the Kabat position H58 is Tyr,the amino acid at position 11 of SEQ ID NO:158 which is the amino acidat the Kabat position H59 is Tyr,the amino acid at position 12 of SEQ ID NO:158 which is the amino acidat the Kabat position H60 is Val,the amino acid at position 13 of SEQ ID NO:158 which is the amino acidat the Kabat position H61 is Asp,the amino acid at position 14 of SEQ ID NO:158 which is the amino acidat the Kabat position H62 is Ser,the amino acid at position 15 of SEQ ID NO:158 which is the amino acidat the Kabat position H63 is Val,the amino acid at position 16 of SEQ ID NO:158 which is the amino acidat the Kabat position H64 is Lys andthe amino acid at position 17 of SEQ ID NO:158 which is the amino acidat the Kabat position H65 is Gly.

SEQ ID NO:8 represents a CDR3 consensus sequence for the heavy chain,wherein

the amino acid at position 1 of SEQ ID NO:8 which is the amino acid atthe Kabat position H95 can be Asp or Gly,the amino acid at position 2 of SEQ ID NO:8 which is the amino acid atthe Kabat position H 96 can be Ala or Gly,the amino acid at position 3 of SEQ ID NO:8 which is the amino acid atthe Kabat position H97 can be Ser or Gly,the amino acid at position 4 of SEQ ID NO:8 which is the amino acid atthe Kabat position H98 can be Ser or Arg,the amino acid at position 5 of SEQ ID NO:8 which is the amino acid atthe Kabat position H99 is Trp,the amino acid at position 6 of SEQ ID NO:8 which is the amino acid atthe Kabat position H100 can be Tyr or Ala,the amino acid at position 7 of SEQ ID NO:8 which is the amino acid atthe Kabat position H100a can be Arg or Pro or Asp,the amino acid at position 8 of SEQ ID NO:8 which is the amino acid atthe Kabat position H100b can be Asp or Leu,the amino acid at position 9 of SEQ ID NO:8 which is the amino acid atthe Kabat position H100c can be Trp or Gly or Ala,the amino acid at position 10 of SEQ ID NO:8 which is the amino acid atthe Kabat position H100d can be Phe or Ala,the amino acid at position 11 of SEQ ID NO:8 which is the amino acid atthe Kabat position H100e can be Phe or no amino acid,the amino acid at position 12 of SEQ ID NO:8 which is the amino acid atthe Kabat position H101 is Asp andthe amino acid at position 13 of SEQ ID NO:8 which is the amino acid atthe Kabat position H102 can be Pro or Ile.

SEQ ID NO:159 represents a preferred CDR3 consensus sequence for theheavy chain, wherein

the amino acid at position 1 of SEQ ID NO:159 which is the amino acid atthe Kabat position H95 can be Gly or Asp,the amino acid at position 2 of SEQ ID NO:159 which is the amino acid atthe Kabat position H 96 can be Ala or Gly,the amino acid at position 3 of SEQ ID NO:159 which is the amino acid atthe Kabat position H97 can be Gly or Ser,the amino acid at position 4 of SEQ ID NO:159 which is the amino acid atthe Kabat position H98 can be Arg or Ser,the amino acid at position 5 of SEQ ID NO:159 which is the amino acid atthe Kabat position H99 is Trp,the amino acid at position 6 of SEQ ID NO:159 which is the amino acid atthe Kabat position H100 can be Ala or Tyr,the amino acid at position 7 of SEQ ID NO:159 which is the amino acid atthe Kabat position H100a can be Pro or Arg or Asp,the amino acid at position 8 of SEQ ID NO:159 which is the amino acid atthe Kabat position H100b can be Leu or Asp,the amino acid at position 9 of SEQ ID NO:159 which is the amino acid atthe Kabat position H100c can be Gly or Trp or Ala,the amino acid at position 10 of SEQ ID NO:159 which is the amino acidat the Kabat position H100d can be Ala or Phe,the amino acid at position 11 of SEQ ID NO:159 which is the amino acidat the Kabat position H100e can be Phe or no amino acid,the amino acid at position 12 of SEQ ID NO:159 which is the amino acidat the Kabat position H101 is Asp andthe amino acid at position 13 of SEQ ID NO:159 which is the amino acidat the Kabat position H102 can be Ile or Pro.

SEQ ID NO:160 represents a more preferred CDR3 consensus sequence forthe heavy chain, wherein

the amino acid at position 1 of SEQ ID NO:160 which is the amino acid atthe Kabat position H95 is Gly,the amino acid at position 2 of SEQ ID NO:160 which is the amino acid atthe Kabat position H 96 is Ala,the amino acid at position 3 of SEQ ID NO:160 which is the amino acid atthe Kabat position H97 is Gly,the amino acid at position 4 of SEQ ID NO:160 which is the amino acid atthe Kabat position H98 is Arg,the amino acid at position 5 of SEQ ID NO:160 which is the amino acid atthe Kabat position H99 is Trp,the amino acid at position 6 of SEQ ID NO:160 which is the amino acid atthe Kabat position H100 is Ala,the amino acid at position 7 of SEQ ID NO:160 which is the amino acid atthe Kabat position H100a is Pro,the amino acid at position 8 of SEQ ID NO:160 which is the amino acid atthe Kabat position H100b is Leu,the amino acid at position 9 of SEQ ID NO:160 which is the amino acid atthe Kabat position H100c is Gly,the amino acid at position 10 of SEQ ID NO:160 which is the amino acidat the Kabat position H100d is Ala,the amino acid at position 11 of SEQ ID NO:160 which is the amino acidat the Kabat position H100e is Phe,the amino acid at position 12 of SEQ ID NO:160 which is the amino acidat the Kabat position H101 is Asp andthe amino acid at position 13 of SEQ ID NO:160 which is the amino acidat the Kabat position H102 is Ile.

SEQ ID NO:161 represents a more preferred CDR3 consensus sequence forthe heavy chain, wherein

the amino acid at position 1 of SEQ ID NO:161 which is the amino acid atthe Kabat position H95 is Asp,the amino acid at position 2 of SEQ ID NO:161 which is the amino acid atthe Kabat position H 96 can be Gly or Ala,the amino acid at position 3 of SEQ ID NO:161 which is the amino acid atthe Kabat position H97 can be Ser or Gly,the amino acid at position 4 of SEQ ID NO:161 which is the amino acid atthe Kabat position H98 can be Ser or Arg,the amino acid at position 5 of SEQ ID NO:161 which is the amino acid atthe Kabat position H99 is Trp,the amino acid at position 6 of SEQ ID NO:161 which is the amino acid atthe Kabat position H100 can be Tyr or Ala,the amino acid at position 7 of SEQ ID NO:161 which is the amino acid atthe Kabat position H100a can be Arg or Asp,the amino acid at position 8 of SEQ ID NO:161 which is the amino acid atthe Kabat position H100b can be Asp or Leu,the amino acid at position 9 of SEQ ID NO:161 which is the amino acid atthe Kabat position H100c can be Trp or Ala,the amino acid at position 10 of SEQ ID NO:161 which is the amino acidat the Kabat position H100d is Phe,the amino acid at position 11 of SEQ ID NO:161 which is the amino acidat the Kabat position H101 is Asp andthe amino acid at position 12 of SEQ ID NO:161 which is the amino acidat the Kabat position H102 can be Pro or Ile.

In another embodiment, the polypeptide according to the inventioncomprises as CDRs all three respective consensus CDR sequences asdenoted in SEQ ID NO:6 to 8.

In another embodiment, the polypeptide according to the inventioncomprises at least two of the respective consensus CDR sequences asdenoted in SEQ ID NO:6 to 8.

In another embodiment, the polypeptide according to the inventioncomprises a CDR sequence having at least two of the respective consensussequences as denoted in any of the consensus sequences SEQ ID NOs: 6 to11.

SEQ ID NOs: 9 to 11 represent consensus sequences for CDR regions of thelight chain of an antibody having the ability to bind to SEQ ID NO:2.The consensus sequences are derived from the sequence informationderived from the antibodies, which have been identified by the inventorsto bind specifically to SEQ ID NO:2. In particular, the identifiedconsensus sequences for the CDR regions of the light chain were derivedfrom naturally occurring, human antibodies isolated as described inExample 2A.

SEQ ID NO:9 represents a CDR1 consensus sequence for a kappa light chainimmunoglobulin CDR1 region, wherein

the amino acid at position 1 of SEQ ID NO:9 which is the amino acid atthe Kabat position L24 can be an Arg,the amino acid at position 2 of SEQ ID NO:9 which is the amino acid atthe Kabat position L25 can be Ala or Glu,the amino acid at position 3 of SEQ ID NO:9 which is the amino acid atthe Kabat position L26 can be Ser,the amino acid at position 4 of SEQ ID NO:9 which is the amino acid atthe Kabat position L27 can be Gln,the amino acid at position 5 of SEQ ID NO:9 which is the amino acid atthe Kabat position L28 can be Ser or Gly,the amino acid at position 6 of SEQ ID NO:9 which is the amino acid atthe Kabat position L29 can be Val or Ile,the amino acid at position 7 of SEQ ID NO:9 which is the amino acid atthe Kabat position L30 can be Asn or Arg or Ser,the amino acid at position 8 of SEQ ID NO:9 which is the amino acid atthe Kabat position L31 can be Ser or Asn,the amino acid at position 9 of SEQ ID NO:9 which is the amino acid atthe Kabat position L32 can be Tyr,the amino acid at position 10 of SEQ ID NO:9 which is the amino acid atthe Kabat position L33 can be Leu andthe amino acid at position 11 of SEQ ID NO:9 which is the amino acid atthe Kabat position L34 can be Ala.

SEQ ID NO:10 represents a CDR2 consensus sequence for a kappa lightchain immunoglobulin CDR2 region, wherein

the amino acid at position 1 of SEQ ID NO:10 which is the amino acid atthe Kabat position L50 can be Ala or Gly or Lys or Trp,the amino acid at position 2 of SEQ ID NO:10 which is the amino acid atthe Kabat position L51 can be Val or Ala,the amino acid at position 3 of SEQ ID NO:10 which is the amino acid atthe Kabat position L52 can be Ser or Ala,the amino acid at position 4 of SEQ ID NO:10 which is the amino acid atthe Kabat position L53 can be Thr or Ser or Asn or Ile,the amino acid at position 5 of SEQ ID NO:10 which is the amino acid atthe Kabat position L54 can be Arg or Leu,the amino acid at position 6 of SEQ ID NO:10 which is the amino acid atthe Kabat position L55 can be Ala or Gln or Phe or Glu andthe amino acid at position 7 of SEQ ID NO:10 which is the amino acid atthe Kabat position L56 can be Thr or Ser.

SEQ ID NO:11 represents a CDR3 consensus sequence for a kappa lightchain immunoglobulin CDR3 region, wherein

the amino acid at position 1 of SEQ ID NO:11 which is the amino acid atthe Kabat position L89 can be Gln,the amino acid at position 2 of SEQ ID NO:11 which is the amino acid atthe Kabat position L90 can be Gln,the amino acid at position 3 of SEQ ID NO:11 which is the amino acid atthe Kabat position L91 can be Ala or Tyr,the amino acid at position 4 of SEQ ID NO:11 which is the amino acid atthe Kabat position L92 can be Gly or Asn,the amino acid at position 5 of SEQ ID NO:11 which is the amino acid atthe Kabat position L93 can be Ser,the amino acid at position 6 of SEQ ID NO:11 which is the amino acid atthe Kabat position L94 can be Ser or Phe,the amino acid at position 7 of SEQ ID NO:11 which is the amino acid atthe Kabat position L95 can be Gln or Pro,the amino acid at position 8 of SEQ ID NO:11 which is the amino acid atthe Kabat position L96 can be Gly or Leu andthe amino acid at position 9 of SEQ ID NO:11 which is the amino acid atthe Kabat position L97 can be Thr.

In one embodiment, the polypeptide according to the invention comprisesas CDRs for the light chain all three respective consensus CDR sequencesas denoted in SEQ ID NO:6 to 11.

In one embodiment, the polypeptide according to the invention comprisesat least two CDRs for the light chain selected from all three respectiveconsensus CDR sequences as denoted in SEQ ID NO:9 to 11.

Even more preferred is a polypeptide comprising at least two CDRsequences selected from the consensus CDR sequences denoted for thelight chain (SEQ ID NO:9 to 11) or at least two of the consensus CDRsequences denoted for the heavy chain (SEQ ID NO:6 to 8) or at least oneCDR from the light chain (SEQ ID NO:9 to 11) and at least one CDR fromthe heavy chain (SEQ ID NO:6 to 8).

In one embodiment, the polypeptide according to the invention comprisesas CDRs for the light chain all three respective consensus CDR sequencesas denoted in SEQ ID NO:9 to 11.

Even more preferred is a polypeptide comprising at least two CDRsequences selected from the consensus CDR sequences denoted for thelight chain (SEQ ID NO:9 to 11) and/or at least two of the consensus CDRsequences denoted for the heavy chain (SEQ ID NO:6 to 8).

In one embodiment, isolated, monoclonal, anti-β-amyloid antibodies areprovided that comprise more than one amino acid sequence selected fromat least two consensus amino acid sequences of the group consisting ofSEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10and SEQ ID NO: 11, wherein each of said more than one amino acidsequence is from a different SEQ ID NO, wherein said antibody binds todimeric forms of Aβ with higher affinity than to monomeric forms of Aβ.

In a further embodiment, the polypeptide according to the inventioncomprises as CDR1 on the heavy chain one of the sequences as denoted inSEQ ID NOs: 13 to 20, as CDR2 on the heavy chain one of the sequence asdenoted in SEQ ID NOs: 21 to 27 and 149, and/or as CDR3 on the heavychain one of the sequences as denoted in SEQ ID NOs: 28 to 32.

In another embodiment the polypeptide according to the inventioncomprises at least two consensus CDR sequences selected from consensussequences for CDR 1, CDR 2 and CDR 3 of the heavy chain and CDR 1, CDR 2and CDR 3 of the light chain, wherein the consensus sequences arederived by aligning the sequences of the following antibody variableregions according to the Kabat numbering:

-   -   a) for CDR1 of the heavy chain, SEQ ID NOs: 56 to 71 and 148    -   b) for CDR2 of the heavy chain, SEQ ID NOs: 56 to 71 and 148    -   c) for CDR3 of the heavy chain, SEQ ID NOs: 56 to 71 and 148    -   d) for CDR1 of the light chain, SEQ ID NOs: 47 to 55 and 145 to        147    -   e) for CDR2 of the light chain SEQ ID NOs: 47 to 55 and 145 to        147    -   f) for CDR3 of the light chain, SEQ ID Nos: 47 to 55 and 145 to        147

SEQ ID NOs 13 to 20 represent the CDR1 sequences, SEQ ID NOs 21 to 27and 149 represent the CDR2 sequences and SEQ ID NOs 28 to 32 representthe CDR3 sequences found by the inventors to be present on the heavychain of antibodies binding to a C-terminal part of Aβ(1-40), inparticular to Aβ(21-37).

In a further embodiment, the polypeptide according to the presentinvention comprises as CDR1 on the light chain one of the sequences asdenoted in SEQ ID NOs: 33 to 37, as CDR2 on the light chain one of thesequence as denoted in SEQ ID NOs: 38 to 43 and 150 to 152, and/or asCDR3 on the light chain one of the sequences as denoted in SEQ ID NOs:44 to 46.

SEQ ID NOs 33 to 37 represent the CDR1 sequences, SEQ ID NOs 38 to 43and 150 to 152 represent the CDR2 sequences and SEQ ID NOs 44 to 46represent the CDR3 sequences found by the inventors to be present on thelight chain of antibodies binding to a C-terminal part of Aβ(1-40), inparticular to Aβ(21-37).

In a preferred embodiment, a polypeptide according to the presentinvention comprises on the heavy chain a CDR1 sequence selected from SEQID NOs: 13 to 20, a CDR2 sequence selected from SEQ ID NOs: 21 to 27 and148, and/or a CDR3 sequence selected from SEQ ID NOs: 28 to 32, and onthe light chain a CDR1 sequence selected from SEQ ID NOs: 33 to 37, aCDR2 sequence selected from SEQ ID NOs: 38 to 43 and 150 to 152 and/or aCDR3 sequence selected from SEQ ID NOs: 44 to 46.

In particular, a polypeptide according to the present invention cancomprise on the light chain:

-   -   a) as CDR1 SEQ ID NO:34, as CDR2 SEQ ID NO:38 and as CDR3 SEQ ID        NO:44, or    -   b) as CDR1 SEQ ID NO:33, as CDR2 SEQ ID NO:42 and as CDR3 SEQ ID        NO:44, or    -   c) as CDR1 SEQ ID NO:37, as CDR2 SEQ ID NO:43 and as CDR3 SEQ ID        NO:46, or    -   d) as CDR1 SEQ ID NO:34, as CDR2 SEQ ID NO:40 and as CDR3 SEQ ID        NO:44, or    -   e) as CDR1 SEQ ID NO:35, as CDR2 SEQ ID NO:38 and as CDR3 SEQ ID        NO:44, or    -   f) as CDR1 SEQ ID NO:33, as CDR2 SEQ ID NO:41 and as CDR3 SEQ ID        NO:44, or    -   g) as CDR1 SEQ ID NO:33, as CDR2 SEQ ID NO:41 and as CDR3 SEQ ID        NO:45, or    -   h) as CDR1 SEQ ID NO:34, as CDR2 SEQ ID NO:38 and as CDR3 SEQ ID        NO:44, or    -   i) as CDR1 SEQ ID NO:36, as CDR2 SEQ ID NO:39 and as CDR3 SEQ ID        NO:44.

In particular, a polypeptide according to the present invention can alsocomprise on the heavy chain:

-   -   a) as CDR1 SEQ ID NO:13, as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID        NO:28, or    -   b) as CDR1 SEQ ID NO:14, as CDR2 SEQ ID NO:27 and as CDR3 SEQ ID        NO:30, or    -   c) as CDR1 SEQ ID NO:13, as CDR2 SEQ ID NO:26 and as CDR3 SEQ ID        NO:28, or    -   d) as CDR1 SEQ ID NO:14, as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID        NO:30, or    -   e) as CDR1 SEQ ID NO:15, as CDR2 SEQ ID NO:23 and as CDR3 SEQ ID        NO:29, or    -   f) as CDR1 SEQ ID NO:15, as CDR2 SEQ ID NO:22 and as CDR3 SEQ ID        NO:29, or    -   g) as CDR1 SEQ ID NO:20, as CDR2 SEQ ID NO:27 and as CDR3 SEQ ID        NO:31, or    -   h) as CDR1 SEQ ID NO:18, as CDR2 SEQ ID NO:25 and as CDR3 SEQ ID        NO:31, or    -   i) as CDR1 SEQ ID NO:18, as CDR2 SEQ ID NO:27 and as CDR3 SEQ ID        NO:31, or    -   j) as CDR1 SEQ ID NO:14, as CDR2 SEQ ID NO:27 and as CDR3 SEQ ID        NO:31, or    -   k) as CDR1 SEQ ID NO:14, as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID        NO:31, or    -   l) as CDR1 SEQ ID NO:16, as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID        NO:31, or    -   m) as CDR1 SEQ ID NO:19, as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID        NO:32, or    -   n) as CDR1 SEQ ID NO:16, as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID        NO:28, or    -   o) as CDR1 SEQ ID NO:17, as CDR2 SEQ ID NO:26 and as CDR3 SEQ ID        NO:31, or    -   p) as CDR1 SEQ ID NO:14, as CDR2 SEQ ID NO:24 and as CDR3 SEQ ID        NO:28.

Even more preferred is a polypeptide according to the present inventioncomprising:

-   -   a) on the light chain as CDR1 SEQ ID NO:34, as CDR2 SEQ ID NO:38        and as CDR3 SEQ ID NO:44, and on the heavy chain as CDR1 SEQ ID        NO:13, as CDR2 SEQ ID NO:21 and as CDR3 SEQ ID NO:28, or    -   b) on the light chain as CDR1 SEQ ID NO:33, as CDR2 SEQ ID NO:42        and as CDR3 SEQ ID NO:44, and on the heavy chain as CDR1 SEQ ID        NO:14, as CDR2 SEQ ID NO:27 and as CDR3 SEQ ID NO:30.

Even more preferred is a polypeptide according to the present inventioncomprising a light variable chain having the sequence of SEQ ID NO:53and a variable heavy chain having the sequence of SEQ ID NO:60, or apolypeptide comprising a light chain having the CDR sequences of SEQ IDNO:33, SEQ ID NO: 41 and SEQ ID NO:45 and a heavy chain having the CDRsequences of SEQ ID NO:15, SEQ ID NO:23 and SEQ ID NO:29.

SEQ ID NOs: 47 to 55 and 145 to 147 denote variable regions of the lightchain and SEQ ID NOs: 56 to 71 and 148 denote variable regions of theheavy chain of antibodies having the ability to bind specifically to aC-terminal part of Aβ(1-40), in particular to Aβ(21-37), as determinedby the inventors. The variable regions of the heavy and light chain areresponsible for antigen specificity. Therefore, in a further embodiment,the polypeptide according to the present invention comprises a sequenceselected from SEQ ID NOs: 47 to 55 and 145 to 147 and/or a sequenceselected from SEQ ID NOs: 56 to 71 and 148. In a particularly preferredembodiment the polypeptide according to the present invention comprisesSEQ ID NO:47 and SEQ ID NO:56 or SEQ ID NO:48 and SEQ ID NO:57.

Each of the light chains of SEQ ID NOs: 47 to 55 and 145 to 147 can becombined with any of the heavy chains of SEQ ID NOs: 56 to 71 and 148.Accordingly, anti-β-amyloid antibodies are provided that comprise SEQ IDNO: 47 and SEQ ID NO:56; SEQ ID NO: 47 and SEQ ID NO:57; SEQ ID NO: 47and SEQ ID NO: 58; SEQ ID NO: 47 and SEQ ID NO: 59; SEQ ID NO: 47 andSEQ ID NO:60; SEQ ID NO: 47 and SEQ ID NO: 61; SEQ ID NO: 47 and SEQ IDNO: 62; SEQ ID NO: 47 and SEQ ID NO: 63; SEQ ID NO: 47 and SEQ ID NO:64; SEQ ID NO: 47 and SEQ ID NO: 65; SEQ ID NO: 47 and SEQ ID NO: 66;SEQ ID NO: 47 and SEQ ID NO: 67; SEQ ID NO: 47 and SEQ ID NO: 68; SEQ IDNO: 47 and SEQ ID NO: 69; SEQ ID NO: 47 and SEQ ID NO: 70; SEQ ID NO: 47and SEQ ID NO: 71; SEQ ID NO: 47 and SEQ ID NO: 148; SEQ ID NO: 48 andSEQ ID NO:56; SEQ ID NO: 48 and SEQ ID NO:57; SEQ ID NO: 48 and SEQ IDNO:58; SEQ ID NO: 48 and SEQ ID NO:59; SEQ ID NO: 48 and SEQ ID NO:60;SEQ ID NO: 48 and SEQ ID NO:61; SEQ ID NO: 48 and SEQ ID NO:62; SEQ IDNO: 48 and SEQ ID NO:63; SEQ ID NO: 48 and SEQ ID NO:64; SEQ ID NO: 48and SEQ ID NO:65; SEQ ID NO: 48 and SEQ ID NO:66; SEQ ID NO: 48 and SEQID NO:67; SEQ ID NO: 48 and SEQ ID NO:68; SEQ ID NO: 48 and SEQ IDNO:69; SEQ ID NO: 48 and SEQ ID NO:70; SEQ ID NO: 48 and SEQ ID NO:71;SEQ ID NO: 48 and SEQ ID NO:148; SEQ ID NO: 49 and SEQ ID NO:56; SEQ IDNO: 49 and SEQ ID NO:57; SEQ ID NO: 49 and SEQ ID NO: 58; SEQ ID NO: 49and SEQ ID NO: 59; SEQ ID NO: 49 and SEQ ID NO: 60; SEQ ID NO: 49 andSEQ ID NO: 61; SEQ ID NO: 49 and SEQ ID NO: 62; SEQ ID NO: 49 and SEQ IDNO: 63; SEQ ID NO: 49 and SEQ ID NO: 64; SEQ ID NO: 49 and SEQ ID NO:65; SEQ ID NO: 49 and SEQ ID NO: 66; SEQ ID NO: 49 and SEQ ID NO: 67;SEQ ID NO: 49 and SEQ ID NO: 68; SEQ ID NO: 49 and SEQ ID NO: 69; SEQ IDNO: 49 and SEQ ID NO: 70; SEQ ID NO: 49 and SEQ ID NO: 71; SEQ ID NO: 49and SEQ ID NO: 148; SEQ ID NO: 50 and SEQ ID NO: 56; SEQ ID NO: 50 andSEQ ID NO: 57; SEQ ID NO: 50 and SEQ ID NO: 58; SEQ ID NO: 50 and SEQ IDNO: 59; SEQ ID NO: 50 and SEQ ID NO: 60; SEQ ID NO: 50 and SEQ ID NO:61; SEQ ID NO: 50 and SEQ ID NO: 62; SEQ ID NO: 50 and SEQ ID NO: 63;SEQ ID NO: 50 and SEQ ID NO: 64; SEQ ID NO: 50 and SEQ ID NO: 65; SEQ IDNO: 50 and SEQ ID NO: 66; SEQ ID NO: 50 and SEQ ID NO: 67; SEQ ID NO: 50and SEQ ID NO: 68; SEQ ID NO: 50 and SEQ ID NO: 69; SEQ ID NO: 50 andSEQ ID NO: 70; SEQ ID NO: 50 and SEQ ID NO: 71; SEQ ID NO: 50 and SEQ IDNO: 148; SEQ ID NO: 51 and SEQ ID NO: 56; SEQ ID NO: 51 and SEQ ID NO:57; SEQ ID NO: 51 and SEQ ID NO: 58; SEQ ID NO: 51 and SEQ ID NO: 59;SEQ ID NO: 51 and SEQ ID NO: 60; SEQ ID NO: 51 and SEQ ID NO: 61; SEQ IDNO: 51 and SEQ ID NO: 62; SEQ ID NO: 51 and SEQ ID NO: 63; SEQ ID NO: 51and SEQ ID NO: 64; SEQ ID NO: 51 and SEQ ID NO: 65; SEQ ID NO: 51 andSEQ ID NO: 66; SEQ ID NO: 51 and SEQ ID NO: 67; SEQ ID NO: 51 and SEQ IDNO: 68; SEQ ID NO: 51 and SEQ ID NO: 69; SEQ ID NO: 51 and SEQ ID NO:70; SEQ ID NO: 51 and SEQ ID NO: 71; SEQ ID NO: 51 and SEQ ID NO: 148;SEQ ID NO: 52 and SEQ ID NO: 56; SEQ ID NO: 52 and SEQ ID NO: 57; SEQ IDNO: 52 and SEQ ID NO: 58; SEQ ID NO: 52 and SEQ ID NO: 59; SEQ ID NO: 52and SEQ ID NO: 60; SEQ ID NO: 52 and SEQ ID NO: 61; SEQ ID NO: 52 andSEQ ID NO: 62; SEQ ID NO: 52 and SEQ ID NO: 63; SEQ ID NO: 52 and SEQ IDNO: 64; SEQ ID NO: 52 and SEQ ID NO: 65; SEQ ID NO: 52 and SEQ ID NO:66;SEQ ID NO: 52 and SEQ ID NO: 67; SEQ ID NO: 52 and SEQ ID NO: 68; SEQ IDNO: 52 and SEQ ID NO: 69; SEQ ID NO: 52 and SEQ ID NO: 70; SEQ ID NO: 52and SEQ ID NO: 71; SEQ ID NO: 52 and SEQ ID NO:148; SEQ ID NO: 53 andSEQ ID NO: 56; SEQ ID NO: 53 and SEQ ID NO: 57; SEQ ID NO: 53 and SEQ IDNO: 58; SEQ ID NO: 53 and SEQ ID NO: 59; SEQ ID NO: 53 and SEQ ID NO:60; SEQ ID NO: 53 and SEQ ID NO: 61; SEQ ID NO: 53 and SEQ ID NO: 62;SEQ ID NO: 53 and SEQ ID NO: 63; SEQ ID NO: 53 and SEQ ID NO: 64; SEQ IDNO: 53 and SEQ ID NO: 65; SEQ ID NO: 53 and SEQ ID NO: 66; SEQ ID NO: 53and SEQ ID NO: 67; SEQ ID NO: 53 and SEQ ID NO: 68; SEQ ID NO: 53 andSEQ ID NO: 69; SEQ ID NO: 53 and SEQ ID NO: 70; SEQ ID NO: 53 and SEQ IDNO: 71; SEQ ID NO: 53 and SEQ ID NO: 148; SEQ ID NO: 54 and SEQ ID NO:56; SEQ ID NO: 54 and SEQ ID NO: 57; SEQ ID NO: 54 and SEQ ID NO: 58;SEQ ID NO: 54 and SEQ ID NO: 59; SEQ ID NO: 54 and SEQ ID NO: 60; SEQ IDNO: 54 and SEQ ID NO: 61; SEQ ID NO: 54 and SEQ ID NO: 62; SEQ ID NO: 54and SEQ ID NO: 63; SEQ ID NO: 54 and SEQ ID NO: 64; SEQ ID NO: 54 andSEQ ID NO: 65; SEQ ID NO: 54 and SEQ ID NO: 66; SEQ ID NO: 54 and SEQ IDNO: 67; SEQ ID NO: 54 and SEQ ID NO: 68; SEQ ID NO: 54 and SEQ ID NO:69; SEQ ID NO: 54 and SEQ ID NO: 70; SEQ ID NO: 54 and SEQ ID NO: 71;SEQ ID NO: 54 and SEQ ID NO: 148; SEQ ID NO: 55 and SEQ ID NO: 56; SEQID NO: 55 and SEQ ID NO: 57; SEQ ID NO: 55 and SEQ ID NO: 58; SEQ ID NO:55 and SEQ ID NO: 59; SEQ ID NO: 55 and SEQ ID NO: 60; SEQ ID NO: 55 andSEQ ID NO: 61; SEQ ID NO: 55 and SEQ ID NO: 62; SEQ ID NO: 55 and SEQ IDNO: 63; SEQ ID NO: 55 and SEQ ID NO: 64; SEQ ID NO: 55 and SEQ ID NO:65; SEQ ID NO: 55 and SEQ ID NO: 66; SEQ ID NO: 55 and SEQ ID NO: 67;SEQ ID NO: 55 and SEQ ID NO: 68; SEQ ID NO: 55 and SEQ ID NO: 69; SEQ IDNO: 55 and SEQ ID NO: 70; SEQ ID NO: 55 and SEQ ID NO: 71; SEQ ID NO: 55and SEQ ID NO: 148; SEQ ID NO: 145 and SEQ ID NO: 56; SEQ ID NO: 145 andSEQ ID NO: 57; SEQ ID NO: 145 and SEQ ID NO: 58; SEQ ID NO: 145 and SEQID NO: 59; SEQ ID NO: 145 and SEQ ID NO: 60; SEQ ID NO: 145 and SEQ IDNO: 61; SEQ ID NO: 145 and SEQ ID NO: 62; SEQ ID NO: 145 and SEQ ID NO:63; SEQ ID NO: 145 and SEQ ID NO: 64; SEQ ID NO: 145 and SEQ ID NO: 65;SEQ ID NO: 145 and SEQ ID NO: 66; SEQ ID NO: 145 and SEQ ID NO: 67; SEQID NO: 145 and SEQ ID NO: 68; SEQ ID NO: 145 and SEQ ID NO: 69; SEQ IDNO: 145 and SEQ ID NO: 70; SEQ ID NO: 145 and SEQ ID NO: 71; SEQ ID NO:145 and SEQ ID NO: 148; SEQ ID NO: 146 and SEQ ID NO: 56; SEQ ID NO: 146and SEQ ID NO: 57; SEQ ID NO: 146 and SEQ ID NO: 58; SEQ ID NO: 146 andSEQ ID NO: 59; SEQ ID NO: 146 and SEQ ID NO: 60; SEQ ID NO: 146 and SEQID NO: 61; SEQ ID NO: 146 and SEQ ID NO: 62; SEQ ID NO: 146 and SEQ IDNO: 63; SEQ ID NO: 146 and SEQ ID NO: 64; SEQ ID NO: 146 and SEQ ID NO:65; SEQ ID NO: 146 and SEQ ID NO: 66; SEQ ID NO: 146 and SEQ ID NO: 67;SEQ ID NO: 146 and SEQ ID NO: 68; SEQ ID NO: 146 and SEQ ID NO: 69; SEQID NO: 146 and SEQ ID NO: 70; SEQ ID NO: 146 and SEQ ID NO: 71; SEQ IDNO: 146 and SEQ ID NO: 148; SEQ ID NO: 147 and SEQ ID NO: 56; SEQ ID NO:147 and SEQ ID NO: 57; SEQ ID NO: 147 and SEQ ID NO: 58; SEQ ID NO: 147and SEQ ID NO: 59; SEQ ID NO: 147 and SEQ ID NO: 60; SEQ ID NO: 147 andSEQ ID NO: 61; SEQ ID NO: 147 and SEQ ID NO: 62; SEQ ID NO: 147 and SEQID NO: 63; SEQ ID NO: 147 and SEQ ID NO: 64; SEQ ID NO: 147 and SEQ IDNO: 65; SEQ ID NO: 147 and SEQ ID NO: 66; SEQ ID NO: 147 and SEQ ID NO:67; SEQ ID NO: 147 and SEQ ID NO: 68; SEQ ID NO: 147 and SEQ ID NO: 69;SEQ ID NO: 147 and SEQ ID NO: 70; SEQ ID NO: 147 and SEQ ID NO: 71; orSEQ ID NO: 147 and SEQ ID NO: 148;

In preferred embodiments, the anti-β-amyloid antibodies comprise SEQ IDNO: 145 and SEQ ID NO:60; SEQ ID NO: 53 and SEQ ID NO:60; SEQ ID NO: 51and SEQ ID NO:60; SEQ ID NO: 52 and SEQ ID NO:60; SEQ ID NO: 146 and SEQID NO:60; SEQ ID NO: 53 and SEQ ID NO:148; SEQ ID NO: 55 and SEQ IDNO:61; SEQ ID NO: 145 and SEQ ID NO:62; SEQ ID NO: 54 and SEQ ID NO:60;SEQ ID NO: 54 and SEQ ID NO:61; SEQ ID NO: 54 and SEQ ID NO:148; SEQ IDNO: 145 and SEQ ID NO:148; or SEQ ID NO: 146 and SEQ ID NO:61.

In addition, SEQ ID NOs 72 to 74 denote constant regions of the lightchain and SEQ ID NOs 75 to 77 denote constant regions of the heavy chainof antibodies having the ability to bind specifically to a C-terminalpart of Aβ(1-40), in particular to Aβ(21-37). Therefore, in a furtherembodiment, the polypeptide according to the present invention cancomprise a sequence selected from SEQ ID NOs: 72 to 74 and/or a sequenceselected from SEQ ID NOs: 75 to 77. In particular, the polypeptideaccording to the present invention can comprise a sequence selected fromSEQ ID NO:72 or 73 and a sequence selected from SEQ ID NOs 75 to 77.

The complete sequences, consisting of variable region and constantregion, for the light chain of antibodies having the ability to bindspecifically to a C-terminal part of Aβ(1-40), in particular toAβ(21-37), are denoted in SEQ ID NOs: 78 to 93.

Therefore, in another embodiment of the present invention, thepolypeptide according to the present invention comprises a sequenceselected from SEQ ID NOs: 78 to 93.

The complete sequences, consisting of variable region and constantregion, for the heavy chain of antibodies having the ability to bindspecifically to a C-terminal part of Aβ(1-40), in particular toAβ(21-37), are denoted in SEQ ID NOs: 94 to 137. Therefore, in anotherembodiment of the present invention, the polypeptide according to thepresent invention comprises a sequence selected from SEQ ID NOs: 94 to137.

In an even more preferred embodiment, the polypeptide according to thepresent invention comprises a sequence as denoted in SEQ ID NO:78 and asequence as denoted in SEQ ID NO:94 or a sequence as denoted in SEQ IDNO:80 and a sequence as denoted in SEQ ID NO:97. The same applies to theanalogous isoforms of respective chains and combinations thereof (as canbe taken from FIG. 23 a).

Consensus sequences for framework regions also can be identified. If theamino acid sequences of the variable regions of the heavy and lightchains are aligned respectively according to the Kabat rules, theso-called framework regions N-terminal to CDR1, between CDR1 and CDR2,between CDR2 and CDR3 and C-terminal to CDR3 can be compared in a wayanalogous to that described above for the CDR regions, and consensussequences for the framework of the antibodies of the invention can bederived.

It has to be noted that the inventors found that the N-terminalsequence, about 18 amino acid residues, of kappa light chain ofantibodies having the ability to bind specifically to a C-terminal partof Aβ(1-40), in particular to Aβ(21-37), is well conserved. This appliesfor antibodies derived from IVIgG preparations as well as for antibodiesderived from patient serum. In FIG. 31 said sequences are depicted,additionally also denoted as SEQ ID NOs: 138 to 143. It is contemplated,that the conservative nature of the N-terminus of kappa light chain ofthese antibodies might contribute to antigen specificity and/orprevention of plaque formation when bound to Aβ peptide. Thus, in apreferred embodiment, the polypeptide according to the present inventioncomprises a sequence as denoted in the consensus sequence of SEQ ID NO:44, in particular a sequence as denoted in SEQ ID NOs: 138 to 143.

In another embodiment, the inventive polypeptides bind specifically tooligomeric forms of β-amyloid polypeptide. By way of non-limitingexample, the polypeptides can bind oligomeric forms of Aβ(1-40) oroligomeric fauns of Aβ(12-20) or oligomeric forms of Aβ(21-37). In oneaspect, the inventive polypeptides are capable of binding specificallyto oligomeric fauns of Aβ(1-40) when incubated overnight at 10 mM sodiumphosphate, 150 mM NaCl, pH 7.4 at 4° C.

In another aspect, the inventive autoantibodies bind with a higheraffinity to dimers of Aβ than to corresponding monomers of Aβ.

In general, binding affinity is a measure of antibody-antigencombination and concerns the selectivity with which a given antibodybinds to an epitope when compared with binding to any other epitope.This preferential or selective binding can be quantified as a bindingaffinity or titer. Methods of calculating antibody affinity arewell-known in the field. See, e.g. Practical Immunology Ch. 3, Frank C.Hay & Olwyn M. R. Westwood, Blackwell Publishing (2002); MeasuringImmunity: Basic Biology and Clinical Assessment, Ch. 16, Michael T.Lotze & Angus W. Thomson (eds.), Academic Press (2005), both which areincorporated herein by reference.

In a preferred embodiment, where the polypeptide according to theinvention is an antibody, said antibody may be a monoclonal antibody.Monoclonal antibodies have the advantage that they exhibit less crossreactivity.

In another preferred embodiment the polypeptides according to thepresent invention comprises derivatives and/or fragments of antibodiesbinding to Aβ(21-37) (SEQ ID NO:2). It is well known in the art thatantibodies can be treated enzymatically, e.g. with proteases in order toobtain fragments of antibodies which retain the antigen bindingproperties of the intact antibody, but which lack the Fc-domain. Suchfragments are for example Fab or F(ab′)₂ fragments, which are obtainablevia enzymatic digest of antibodies with papain or pepsin protease,respectively. Another fragment of an antibody is a single chain variablefragment (scFv) of an antibody, i.e. a fusion of the variable regions ofthe heavy and light chains of an antibody via a short flexible linker(usually serine, glycine). Normally, such a chimeric molecule retainsthe specificity of the original antibody, despite removal of theconstant regions and the introduction of a linker peptide. Single chainvariable fragments can be obtained by genetic engineering of arespective nucleic acid encoding for such a fusion protein. Theadvantage of such a fragment is that it consists only of a singlepolypeptide chain which is more easily expressed and properly folded inartificial expression systems than the whole antibody, which comprisesat least 4 polypeptide chains which need a correct assembly in order tofunction adequately.

If the polypeptide according the invention is an antibody, a humanantibody is preferred due to its low immunogenicity in humans. However,the antibody or fragment thereof can be derived from any speciessuitable for antibody production. Non-human antibodies can be derived inparticular from mouse, chicken, rabbit, rat, donkey, camel, dromedary,shark and llamas. Antibodies of camel, dromedary, shark and llamas havethe advantage that these animals have antibodies which consist only of ahomodimer. Thus, such polypeptides have similar advantages in expressionand assembly as described above for single chain variable fragments.

In another preferred embodiment the antibody, derivatives or fragmentsthereof is a humanized antibody, derivative or fragment thereof, i.e.while the antigen binding domain or parts thereof is/are of non-humanorigin, the rest of the antibody, derivative or fragment thereof is ofhuman origin. In another preferred embodiment the antibody, derivativeor fragment thereof is chimeric, i.e. while the variable domain or partsthereof is/are of non-human origin the constant domain is of humanorigin. Both embodiments serve the purpose to reduce negative sideeffects due to immunogenic properties of protein domains of non-humanorigin. In an even more preferred embodiment, the polypeptide binding toepitope Aβ(21-37) (SEQ ID NO:2) of amyloid-beta peptide (1-40) (SEQ IDNO:1) is an antibody derivative or fragment which comprises only theparatope of an antibody binding to said epitope. Example for such aparatope is, for example, a polypeptide comprising the amino acidsequences for the respective CDR domains of heavy and light chainconnected via the intervening sequences or via synthetic linkers.

In one aspect, the antibodies of the present invention embrace allelicvariants, conservatively modified variants, and minor recombinantmodifications to a specific anti-Aβ(21-37) autoantibody. Amino acidsequence variants of the antibody can be prepared by introducingappropriate nucleotide changes into the encoding DNA, or by peptidesynthesis. Such variants include, for example, deletions and/orinsertions and/or substitutions of residues within the amino acidsequences of the antibodies. Any combination of deletion, insertion, andsubstitution is made to arrive at the final construct, provided that thefinal construct possesses the desired characteristics. The amino acidchanges also may alter post-translational processes of antibody, such aschanging the number or position of glycosylation sites.

Amino acid sequence insertions can include, for example, amino- and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintra-sequence insertions of single or multiple amino acid residues.Examples of terminal insertions include an antibody with an N-terminalmethionyl residue or the specific binding agent or antibody (includingantibody fragment) fused to an epitope tag or a salvage receptorepitope. Other insertional variants include a fusion to a polypeptidewhich increases the serum half-life of the antibody, e.g. at theN-terminus or C-terminus.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculeremoved and a different residue inserted in its place. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: cys, ser, thr;    -   (3) acidic: asp, glu;    -   (4) basic: asn, gin, his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Conservative substitutions involve replacing an amino acid with anothermember of its class. Non-conservative substitutions involve replacing amember of one of these classes with a member of another class. Exampleof conservative substitutions include replacing ala with val, leu orile; arg with lys, gin or asn; asn with gln, his, asp, lys, or gln; aspwith glu or asn; cys with ser or ala; gln with asn or glu; glu with aspor gin; gly with ala; his with asn, gin, lys, or arg; ile with leu, val,met, ala or phe; leu with norleucine, ile, val, met, ala, or phe; lyswith arg, gly, asn; met with leu, phe, or ile; phe with leu, val, ile,ala, or tyr; pro with ala; ser with thr; thr with ser; trp with tyr orphe; tyr with trp, phe, thr or ser; and val with ile, leu, met, phe,ala, or norleucine.

Any cysteine residue not involved in maintaining the proper conformationof the specific binding agent or humanized or variant antibody also maybe substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking. Conversely,cysteine bond(s) may be added to the specific binding agent or antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

Altered glycosylation variants also can be produced that have a modifiedglycosylation pattern relative to the parent antibody, for example, bydeleting one or more carbohydrate moieties found in the antibody, and/oradding one or more glycosylation sites that are not present in theantibody. Glycosylation of polypeptides including antibodies istypically either N-linked or O-linked. N-linked refers to the attachmentof the carbohydrate moiety to the side chain of an asparagine residue.The tripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. The presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. Thus, N-linkedglycosylation sites may be added to an antibody by altering the aminoacid sequence such that it contains one or more of these tripeptidesequences. O-linked glycosylation refers to the attachment of one of thesugars N-aceylgalactosamine, galactose, or xylose to a hydroxyaminoacid, most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine also may be used. O-linked glycosylation sites may beadded to a specific binding agent or antibody by inserting orsubstituting one or more serine or threonine residues to the sequence ofthe antibody.

Modifications to increase serum half-life also may desirable, forexample, by incorporation of or addition of a salvage receptor bindingepitope (e.g., by mutation of the appropriate region or by incorporatingthe epitope into a peptide tag that is then fused to the antibody ateither end or in the middle, e.g., by DNA or peptide synthesis; See,e.g., WO 96/32478) or adding molecules such as PEG or other watersoluble polymers, including polysaccharide polymers.

Preparation of Antibodies Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. Alternatively, antigen may be injected directlyinto the animal's lymph node (see Kilpatrick et al., Hybridoma,16:381-389, 1997). An improved antibody response may be obtained byconjugating the relevant antigen to a protein that is immunogenic in thespecies to be immunized, e.g., keyhole limpet hemocyanin, serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctionalor derivatizing agent, for example, maleimidobenzoyl sulfosuccinimideester (conjugation through cysteine residues), N-hydroxysuccinimide(through lysine residues), glutaraldehyde, succinic anhydride or otheragents known in the art.

Animals are immunized against the antigen, immunogenic conjugates orderivatives by combining, e.g., 100 μg of the protein or conjugate (formice) with 3 volumes of Freund's complete adjuvant and injecting thesolution intradermally at multiple sites. One month later, the animalsare boosted with ⅕ to 1/10 the original amount of peptide or conjugatein Freund's complete adjuvant by subcutaneous injection at multiplesites. At 7-14 days post-booster injection, the animals are bled and theserum is assayed for antibody titer Animals are boosted until the titerplateaus. Preferably, the animal is boosted with the conjugate of thesame antigen, but conjugated through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

Monoclonal Antibodies

Monoclonal antibodies can be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or by recombinantDNA methods. In the hybridoma method, a mouse or other appropriate hostanimal, such as rats, hamster or macaque monkey, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells and are sensitive to a medium. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Exemplary murine myeloma lines include those derived from MOP-21 andM.C.-11 mouse tumors available from the Salk Institute Cell DistributionCenter, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells availablefrom the American Type Culture Collection, Rockville, Md. USA. Culturemedium in which hybridoma cells are growing is assayed for production ofmonoclonal antibodies directed against the antigen. Preferably, thebinding specificity of monoclonal antibodies produced by hybridoma cellsis determined by immunoprecipitation or by an in vitro binding assay,such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay(ELISA). The binding affinity of the monoclonal antibody can bedetermined, for example, by BIAcore or Scatchard analysis (Munson etal., Anal. Biochem., 107:220 (1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones can besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEMO or RPMI 1640 medium. In addition, thehybridoma cells can be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography.

Recombinant Production of Antibodies

The amino acid sequence of an immunoglobulin of interest can bedetermined by direct protein sequencing, and suitable encodingnucleotide sequences can be designed according to a universal codontable.

Alternatively, DNA encoding the monoclonal antibodies can be isolatedand sequenced from the hybridoma cells using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of themonoclonal antibodies). Sequence determination will generally requireisolation of at least a portion of the gene or cDNA of interest. Usuallythis requires cloning the DNA or mRNA encoding the monoclonalantibodies. Cloning is carried out using standard techniques (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3,Cold Spring Harbor Press, which is incorporated herein by reference).For example, a cDNA library can be constructed by reverse transcriptionof polyA+ mRNA, preferably membrane-associated mRNA, and the libraryscreened using probes specific for human immunoglobulin polypeptide genesequences. In a preferred embodiment, the polymerase chain reaction(PCR) is used to amplify cDNAs (or portions of full-length cDNAs)encoding an immunoglobulin gene segment of interest (e.g., a light chainvariable segment). The amplified sequences can be cloned readily intoany suitable vector, e.g., expression vectors, minigene vectors, orphage display vectors. It will be appreciated that the particular methodof cloning used is not critical, so long as it is possible to determinethe sequence of some portion of the immunoglobulin polypeptide ofinterest.

One source for RNA used for cloning and sequencing is a hybridomaproduced by obtaining a B cell from the transgenic mouse and fusing theB cell to an immortal cell. An advantage of using hybridomas is thatthey can be easily screened, and a hybridoma that produces a humanmonoclonal antibody of interest selected. Alternatively, RNA can beisolated from B cells (or whole spleen) of the immunized animal. Whensources other than hybridomas are used, it may be desirable to screenfor sequences encoding immunoglobulins or immunoglobulin polypeptideswith specific binding characteristics. One method for such screening isthe use of phage display technology. Phage display is described in e.g.,Dower et al., WO 91/17271, McCafferty et al., WO 92/01047, and Caton andKoprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each ofwhich is incorporated herein by reference. In one embodiment using phagedisplay technology, cDNA from an immunized transgenic mouse (e.g., totalspleen cDNA) is isolated, PCR is used to amplify cDNA sequences thatencode a portion of an immunoglobulin polypeptide, e.g., CDR regions,and the amplified sequences are inserted into a phage vector. cDNAsencoding peptides of interest, e.g., variable region peptides withdesired binding characteristics, are identified by standard techniquessuch as panning.

The sequence of the amplified or cloned nucleic acid is then determined.Typically the sequence encoding an entire variable region of theimmunoglobulin polypeptide is determined, however, sometimes only aportion of a variable region need be sequenced, for example, theCDR-encoding portion. Typically the sequenced portion will be at least30 bases in length, and more often bases coding for at least aboutone-third or at least about one-half of the length of the variableregion will be sequenced.

Sequencing can be carried out on clones isolated from a cDNA library or,when PCR is used, after subcloning the amplified sequence or by directPCR sequencing of the amplified segment. Sequencing is carried out usingstandard techniques (see, e.g., Sambrook et al. (1989) MolecularCloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, andSanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467, whichis incorporated herein by reference). By comparing the sequence of thecloned nucleic acid with published sequences of human immunoglobulingenes and cDNAs, an artisan can determine readily, depending on theregion sequenced, (i) the germline segment usage of the hybridomaimmunoglobulin polypeptide (including the isotype of the heavy chain)and (ii) the sequence of the heavy and light chain variable regions,including sequences resulting from N-region addition and the process ofsomatic mutation. One source of immunoglobulin gene sequence informationis the National Center for Biotechnology Information, National Libraryof Medicine, National Institutes of Health, Bethesda, Md.

Once isolated, the DNA may be operably linked to expression controlsequences or placed into expression vectors, which are then transfectedinto host cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to direct the synthesis of monoclonal antibodiesin the recombinant host cells.

Expression control sequences denote DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome-binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome-binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, operably linkedmeans that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking can be accomplished byligation at convenient restriction sites. If such sites do not exist,synthetic oligonucleotide adaptors or linkers can be used in accordancewith conventional practice.

Cell, cell line, and cell culture are often used interchangeably and allsuch designations include progeny. Transformants and transformed cellsinclude the primary subject cell and cultures derived therefrom withoutregard for the number of transfers. It also is understood that allprogeny may not be precisely identical in DNA content, due to deliberateor inadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included.

Isolated nucleic acids also are provided that encode specificantibodies, optionally operably linked to control sequences recognizedby a host cell, vectors and host cells comprising the nucleic acids, andrecombinant techniques for the production of the antibodies, which maycomprise culturing the host cell so that the nucleic acid is expressedand, optionally, recovering the antibody from the host cell culture orculture medium.

A variety of vectors are known in the art. Vector components can includeone or more of the following: a signal sequence (that, for example, candirect secretion of the antibody), an origin of replication, one or moreselective marker genes (that, for example, can confer antibiotic orother drug resistance, complement auxotrophic deficiencies, or supplycritical nutrients not available in the media), an enhancer element, apromoter, and a transcription termination sequence, all of which arewell known in the art.

Suitable host cells include prokaryote, yeast, or higher eukaryotecells. Suitable prokaryotes include eubacteria, such as Gram-negative orGram-positive organisms, for example, Enterohacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis, Pseudomonas, and Streptomyces. In addition toprokaryotes, eukaryotic microbes such as filamentous fungi or yeast aresuitable cloning or expression hosts for antibody-encoding vectors.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among lower eukaryotic host microorganisms. However, a number ofother genera, species, and strains are commonly available, such asPichia, e.g. P. pastoris, Schizosaccharomyces pombe; Kluyveromyces,Yarrowia; Candida; Trichodemia reesia; Neurospora crassa; Schwanniomycessuch as Schwanniomyces occidentalis; and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts suchas A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodies arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionof such cells are publicly available, e.g., the L-I variant ofAutographa californica NPV and the Bm-5 strain of Bombyx mori NPV.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become routine.Examples of useful mammalian host cell-lines are Chinese hamster ovarycells, including CHOK1 cells (ATCC CCL61) and Chinese hamster ovarycells/−DHFR (DXB-11, DG-44; Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, [Graham et al., J. Gen Virol. 36: 59(1977)]; baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertolicells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidneycells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (WI38, ATCC CCL 75); human hepatomacells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC5 cells and FS4 cells.

The host cells can be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ((DMEM), Sigma) are suitable for culturing the host cells. Inaddition, any of the media described in Ham et al., Meth. Enz. 58: 44(1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO87/00195; or U.S. Pat. Re. No. 30,985 can be used as culture media forthe host cells. Any of these media can be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleotides (such asadenosine and thymidine), antibiotics (such as Gentamycin™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements also can be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the artisan.

The antibody composition can be purified using, for example,hydroxylapatite chromatography, cation or anion exchange chromatography,or preferably affinity chromatography, using the antigen of interest orprotein A or protein G as an affinity ligand. Protein A can be used topurify antibodies that are based on human γ1, γ2, or γ4 heavy chains(Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., 20EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, 25 NJ.) is useful for purification. Other techniques forprotein purification such as ethanol precipitation, Reverse Phase HPLC,chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsopossible depending on the specific binding agent or antibody to berecovered.

A person skilled in the art with the information of this patentapplication at hand will readily know how to perform the methodsaccording to the invention. One example would be to coat a sepharosecolumn with a polypeptide having the sequence of SEQ ID NO: 2, followedby incubation with an immunoglobulin fraction (antibody fraction)derived from the sample, performing a washing step and subsequentelution of the Aβ(21-37) specific antibodies bound to the column. Forthe methods of separation according to the invention the polypeptide,comprising an amino acid sequence of an Aβ polypeptide, wherein thesequence of the Aβ polypeptide has at least the sequence according toSEQ ID NO:2 and at most the sequence according to SEQ ID NO:4,additionally comprises moieties such as tags or markers, in particularbiotin, streptavidin, GST, HIS, STREP-tag, Myc, HA, poly-L-lysine,poly-L-lysine-L-alanine copolymers, poly-Aib (alpha-aminoisobutyricacid), poly-β-alanine, poly-L-alanine, poly-D-lysine,poly-D-lysine-D-alanine copolymers, poly-D-alanine, or combinations ofpoly-L- and D-amino acids. These markers can provide for a facilitatedbinding of this polypeptide to a carrier. Elution, i.e. separation ofpolypeptides according to the present invention from the carrier can,for instance, be achieved by applying a short-term pH change to pH 2, byadding excess of a polypeptide comprising the sequence according to SEQID NO:2 or by increasing the salt content of the elution buffer.Further, non-limiting examples for isolation of polypeptides from theserum of a subject or from commercially available IVIgG preparations aregiven in more detail in the examples section of this application.

Antibodies can not only be derived from a human subject but also from ananimal. Besides isolating pre-existing anti-Aβ(21-37) autoantibodiesfrom the blood of such an animal in analogy to the methods describedabove, such animals can be additionally immunized with a polypeptidecomprising an amino acid sequence of an Aβ polypeptide, wherein thesequence of the Aβ polypeptide has at least the sequence according toSEQ ID NO:2 and at most the sequence according to SEQ ID NO:4. Afterseveral rounds of immunization the corresponding Aβ-specific antibodyproducing B-cells can be obtained from the blood of the animal byroutine methods.

If desired, such B cell clones (of human or animal origin) can beconverted into a cell line, for example by isolating cells from thespleen of an animal and immortalizing them by transfection with EpsteinBarr Virus or by fusing the cells with myeloma cells (hybridomatechnology). The latter is especially useful for producing antibodies inlarge quantities.

Alternatively, the polypeptides of the present invention can be, ifsuitable, directly synthesized, either by conventional polypeptidesynthesis, by in vitro translation or by any other means forsynthesizing polypeptides and proteins. A person skilled in the art willbe familiar with a multitude of appropriate techniques and will bereadily able to apply them to the subject-matter of the presentinvention.

Additionally, the polypeptide binding to the epitope Aβ(21-37) (SEQ IDNO:2) of amyloid-beta (1-40) (SEQ ID NO: 1) can originate from otherproteins than antibodies. As an example, anticalins can be engineered tobind to certain epitopes i.e. to the epitope as denoted in SEQ ID NO: 2.Anticalins are a class of engineered ligand-binding proteins that arebased on the lipocalin scaffold. Using targeted mutagenesis of the loopregion and biochemical selection techniques, variants with novel ligandspecificities, both for low-molecular weight substances and formacromolecular protein targets, can be generated (see DE 199 26 068;Schlehuber et al., J. Mol. Biol. (2000), 297 (5) p. 1105-1120; ExpertOpin Biol Ther. 2005, 5(11), p. 1453-62). Binding of such a polypeptideto an Aβ C-terminal part, in particular to Aβ (21-37) can also providefor an inhibition of polymerization of AD peptide and thus provide anefficient means for treatment and or prophylaxis of Alzheimer's Diseaseand other neurodementing diseases.

The polypeptides according to the invention can also be obtained via arecombinant expression system. In order to express a polypeptideaccording to the invention, a respective nucleic acid expressionconstruct has to be generated. Therefore, the present invention relatesalso to a nucleic acid having a sequence encoding for a polypeptideaccording to the present invention, in particular encoding the lightchain or the heavy chain of one of the above mentioned antibodies,derivatives or fragments thereof or encoding for another proteinaccording to the invention such as an anticalin binding to Aβ(21-37).Such a nucleic acid can be for instance obtained by identifying theamino acid sequence of one of the peptides mentioned above, for instancevia mass spectrometry means, via Edman sequencing or any other methodfor protein sequencing known to the skilled artisan. Following thegenetic code a nucleic acid sequence can be derived from the amino acidsequence. Preferably, the generated nucleic acid sequence is optimizedin regard to the codon usage of the respective expression system ofinterest. Alternatively, cells expressing such an antibody can beisolated (see above) and the genomic loci or mRNA encoding for the heavyand light chain of the antibody specific for the C-Terminus of Aβ aresequenced. For certain expression systems this nucleic acid sequencemight need to be adapted in order to provide for an optimal codon usage.A person skilled in the art with the above mentioned nucleic acidsequences at hand will be readily capable of generating expressionconstructs for use in a suitable expression system. Therefore, thepresent invention also relates to an expression construct providing forthe expression of the polypeptides of the invention and to an isolatedcell, which expresses a polypeptide or a fragment thereof according tothe invention. Expression constructs, i.e. vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses),episomes and artificial chromosomes (e.g., YACs). One of skill in theart would be able to construct a vector by standard recombinanttechniques. Said cell can be for example a myeloma cell, a Chinesehamster ovary cell, a Syrian hamster ovary cell, a human embryonickidney cell, insect cell (baculovirus system), or a transgenic plantcell, which is transformed with an expression vector according to theinvention (see Schillberg et al., Cell Mol Life Sci. 2003 60(3): p.433-445) or a cell, which endogenously expresses a polypeptide accordingto the invention (hybridoma). The expression of recombinant polypeptidesof the invention is not restricted to eukaryotic cells but can also beexpressed in prokaryotes. Recombinant polypeptides according to theinvention can be obtained from any such cell by purification means whichare well known in the art.

In a preferred embodiment, the polypeptide according to the invention isan antibody or a fragment thereof and is optionally encoded by twoexpression vectors, i.e. one expression vector for the light chain andone for the heavy chain.

In order to bind within the polypeptide fragment Aβ(21-37), an antibodyor a fragment thereof requires primarily an intact antigen bindingdomain (variable domain). The constant region of such an antibody isusually not critical for antigen binding. Thus, it is clear for a personskilled in the art, that if the polypeptide according to the inventionis an antibody or a fragment thereof, this antibody/fragment may haveany given isotype selected from the group consisting of IgG, IgM, IgA,IgD and IgE, including all respective subclasses of these isotypes. Ifthe polypeptide according to the present invention is expressed as anantibody in an immune cell or hybridoma cell, the isotype can beswitched by additional expression or administration of activationinduced cytidine deaminase and administration of stimulating factorsknown to the skilled artisan.

In one embodiment, the polypeptides according to the invention arechemically coupled or fused with substances which prevent plaqueaggregation and/or lead to the disintegration of toxic Aβ oligomers.Such a substance could be for example a protease cleaving Amyloid betapolypeptide as used for the experimental characterization of the epitopeaccording to the invention, i.e. Serine-proteases like Trypsin,Chymotrypsin; or Lys-C protease, Arg-C protease, Asp-N-proteases, alsounspecific proteases such as proteinase-K, thermolysine, subtilisin.

The present invention also relates to a method of isolation andseparation of a polypeptide according to the invention from a sample,the method comprising the following steps:

-   -   a) incubating a polypeptide comprising an amino acid sequence of        an Aβ polypeptide, wherein the sequence of the Aβ polypeptide        has at least the sequence according to SEQ ID NO:2, i.e.        Aβ(21-37) and at most the sequence according to SEQ ID NO:4,        i.e. Aβ(12-40), which polypeptide is immobilized on a carrier,        with a sample,    -   b) separating said sample from the carrier.    -   c) separating polypeptides according to the invention bound to        the polypeptide of step a) from the carrier.

In an alternative approach, a polypeptide according to the invention canbe obtained by incubating the sample with the polypeptide comprising anamino acid sequence of an Aβ polypeptide, wherein the sequence of the Aβpolypeptide has at least the sequence according to SEQ ID NO:2 and atmost the sequence according to SEQ ID NO:4 prior to incubation with thecarrier. Thus, the present invention also relates to a method ofseparation of a polypeptide according to the invention from a sample,the method comprising the following steps:

-   -   a) incubating a polypeptide, comprising an amino acid sequence        of an Aβ polypeptide, wherein the sequence of the Aβ polypeptide        has at least the sequence according to SEQ ID NO:2 and at most        the sequence according to SEQ ID NO:4, with a sample and        subsequently    -   b) incubating the sample with a carrier having a binding        affinity for the polypeptide of step a), and    -   c) separating said sample from the carrier, and,    -   d) separating polypeptides according to the invention bound to        the polypeptide of step a) from the carrier.

Strategies and techniques are well known in the art to obtain the abovementioned polypeptides, i.e. via genetic engineering and expression ofthe respective polypeptide in a cell line of interest. Antibodyfragments according to the invention do not have to be physicallyderived from an intact antibody but can also be genetically engineeredby conventional means.

The above mentioned polypeptides can be obtained for example byscreening antibodies derived from the blood of a human subject for thecapacity of binding to amino acid 21 to 37 of SEQ ID NO 1 (i.e. SEQ IDNO: 2).

Methods of Treatment

In another aspect, the present invention relates to the use of the abovementioned polypeptides according to the present invention for use inhuman and veterinary medicine.

In particular, the polypeptides according to the present invention canbe used for the manufacture of a medicament in order to treat and/orprevent the progression of Alzheimer's disease, Down's syndrome,Dementia with Lewy bodies, fronto-temporal dementia, cerebral amyloidangiopathy, and/or amyloidoses.

Furthermore, the present invention relates to the use of a polypeptidecomprising an amino acid sequence of an Aβ peptide, wherein the Aβpeptide has at least the sequence according to SEQ ID NO:2 and at mostthe sequence according to SEQ ID NO:4 for use in human and veterinarymedicine, in particular for the manufacture of a medicament for thetreatment and/or prevention of Alzheimer's disease, Down's syndrome,Dementia with Lewy bodies, fronto-temporal dementia, cerebral amyloidangiopathy, and amyloidoses.

In this invention, the inventors identified two Aβ epitope sequencesrecognized by Aβ autoantibodies isolated from serum of AD patients aswell as of healthy control individuals. The Aβ autoantibodies of healthycontrol individuals were found to specifically recognize a C-terminalpart of the Aβ sequence, namely Aβ(21-37) (SEQ ID NO:2). Furthermore, ADpatients have an increased fraction of antibodies recognizing theAβ(4-10) part of the Aβ polypeptide (SEQ ID NO:3), while having adecreased fraction of the antibodies recognizing the Aβ(21-37) part ofAβ polypeptide. Additionally, the inventors found that in healthyindividuals not suffering from AD, the ratio of the amount of ADautoantibodies directed against a specific C-terminal epitope of Aβ,namely Aβ(21-37) compared to the amount of AD autoantibodies binding toAβ(4-10), is much higher than in AD patients. The binding to thisepitope seems to inhibit the formation of plaques and therefore delaythe onset or progression of AD. This provides the basis for a new,therapeutic approach by administration of agents to a human subject oranimal, which agents bind to said epitope and thereby prevents theaggregation of Aβ peptide. This delays the onset and/or the progressionof Alzheimer's disease and consequently provides a valuabletherapeutic/prophylactic pharmaceutical.

The inventors believe to have identified a class of natural humanantibodies selected over time by evolution to target the toxic oligomersof Aβ as a natural way the human body prevents neurodementing diseases.Such antibodies would be expected not to have pathological effectscaused by the binding to Aβ or related peptides to the brain vessels.Recently, it has been shown that passive immunization with antibodiesdirected against Aβ's N-terminal part causes bleeding in an animaltransgenic mouse model. In these experiments, following 5 months ofpassive immunization, a significant amyloid reduction was found in theneocortex of the immunized mice compared to sham-treated controls.Immunized mice, however, exhibited a more than twofold increase in thefrequency of CAA-associated cerebral hemorrhage in addition to anincrease in hemorrhage severity over controls. These adverse events arebelieved to be caused by Aβ antibodies binding to Aβ deposited in brainvessels (Pfeifer M, et al. 2003. Herzig M C, et al 2004).

Thus, the present invention also relates to the use of the abovementioned polypeptides for the manufacture of a medicament in order totreat and/or prevent the progression of a neurodementing disease,Alzheimer's disease, Down's syndrome, Dementia with Lewy bodies,fronto-temporal dementia, cerebral amyloid angiopathy, and amyloidoses.The treatment/prevention of the above mentioned diseases is provided byprevention of Aβ plaque formation. Depending on the stage of therespective disease this leads to a prevention of the disease (no onsetyet) or to a treatment (after onset of the disease), e.g. by preventingfurther formation of plaques. In a preferred embodiment the medicamentis formulated for the treatment and/or prevention of plaques in thebrain of a patient.

Alternatively, as already indicated above, the present invention relatesalso to the use of a polypeptide comprising an amino acid sequence of anAβ polypeptide, wherein the Aβ polypeptide has at least the sequenceaccording to SEQ ID NO:2 and at most the sequence according to SEQ IDNO:4, for the manufacture of a medicament for the treatment ofAlzheimer's disease, Down's syndrome, Dementia with Lewy bodies,fronto-temporal dementia, cerebral amyloid angiopathy, and/oramyloidoses. In such a scenario, this epitope serves for the activeimmunization of a human or animal subject in order to enhance endogenousantibody production against Aβ(21-37). Such immunization approaches canalso utilize DNA vaccines, which have the benefit of avoiding theadministration of AD protein fragments. Therefore, the present inventionalso relates to a nucleic acid molecule encoding for a polypeptidecomprising an amino acid sequence of an Aβ polypeptide, wherein the Aβpolypeptide has at least the sequence according to SEQ ID NO:2 and atmost the sequence according to SEQ ID NO:4. Consequently the presentinvention also relates to the use of this nucleic acid sequence for themanufacture of a medicament for the treatment and/or prevention ofAlzheimer's disease, Down's syndrome, Dementia with Lewy bodies,fronto-temporal dementia, cerebral amyloid angiopathy or amyloidoses.

Administration and Preparation of Pharmaceutical Formulations

The anti-Aβ antibodies can be formulated into pharmaceuticalcompositions comprising a carrier suitable for the desired deliverymethod. Suitable carriers include any material which, when combined withthe antibody, retains the high-affinity binding of Aβ and is nonreactivewith the subject's immune system. Examples include, but are not limitedto, any of a number of standard pharmaceutical carriers such as sterilephosphate buffered saline solutions, bacteriostatic water, and the like.A variety of aqueous carriers may be used, e.g., water, buffered water,0.4% saline, 0.3% glycine and the like, and can include other proteinsfor enhanced stability, such as albumin, lipoprotein, globulin, etc.,subjected to mild chemical modifications or the like.

Exemplary antibody concentrations in the formulation can range fromabout 0.1 mg/ml to about 180 mg/ml or from about 0.1 mg/mL to about 50mg/mL, or from about 0.5 mg/mL to about 25 mg/mL, or alternatively fromabout 2 mg/mL to about 10 mg/mL. An aqueous formulation of the antibodycan be prepared in a pH-buffered solution, for example, at pH rangingfrom about 4.5 to about 6.5, or from about 4.8 to about 5.5, oralternatively about 5.0. Examples of buffers that are suitable for a pHwithin this range include, for example, acetate (e.g. sodium acetate),succinate (such as sodium succinate), gluconate, histidine, citrate andother organic acid buffers. The buffer concentration can be from about 1mM to about 200 mM, or from about 10 mM to about 60 mM, depending, forexample, on the buffer and the desired isotonicity of the formulation.

A tonicity agent, which also can stabilize the antibody, can be includedin the formulation. Exemplary tonicity agents include sugar alcohols,such as mannitol, sucrose or trehalose. Preferably the aqueousformulation is isotonic, although hypertonic or hypotonic solutions maybe suitable. Exemplary concentrations of the sugar alcohol in theformulation may range from about 1% to about 15% w/v.

A surfactant also may be added to the antibody formulation to reduceaggregation of the formulated antibody and/or minimize the formation ofparticulates in the formulation and/or reduce adsorption. Exemplarysurfactants include nonionic surfactants such as polysorbates (e.g.polysorbate 20, or polysorbate 80) or poloxamers (e.g. poloxamer 188).Exemplary concentrations of surfactant may range from about 0.001% toabout 0.5%, or from about 0.005% to about 0.2%, or alternatively fromabout 0.004% to about 0.01% w/v.

In one embodiment, the formulation contains the above-identified agents(i.e. antibody, buffer, polyol and surfactant) and is essentially freeof one or more preservatives, such as benzyl alcohol, phenol, m-cresol,chlorobutanol and benzethonium Cl. In another embodiment, a preservativemay be included in the formulation, e.g., at concentrations ranging fromabout 0.1% to about 2%, or alternatively from about 0.5% to about 1%.One or more other pharmaceutically acceptable carriers, excipients orstabilizers such as those described in Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980) may be included in theformulation provided that they do not adversely affect the desiredcharacteristics of the formulation. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and include: additional buffering agents; co-solvents;antioxidants including ascorbic acid and methionine; chelating agentssuch as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradablepolymers such as polyesters; and/or salt-forming counter-ions such assodium.

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose,maltose, or dextrins; chelating agents such as EDTA; sugars such assucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions suchas sodium; metal complexes (e.g., Zn-protein complexes); and/ornon-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol(PEG).

In one embodiment, a suitable formulation contains an isotonic buffersuch as a phosphate, acetate or TRIS buffer in combination with atonicity agent such as a sugar alcohol, Sorbitol, sucrose or sodiumchloride which tonicities and stabilizes. One example of such a tonicityagent is 5% Sorbitol or sucrose. In addition, the formulation optionallycan include a surfactant such as to prevent aggregation and forstabilization at 0.01 to 0.02% w/v. The pH of the formulation can rangefrom 4.5-6.5 or 4.5 to 5.5. Other exemplary descriptions ofpharmaceutical formulations for antibodies can be found in US2003/0113316 and U.S. Pat. No. 6,171,586, each incorporated herein byreference in its entirety.

The formulation herein also can contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients also can be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacrylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions or minicells. Such techniques aredisclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).

Suspensions and crystal forms of antibodies are also contemplated.Methods to make suspensions and crystal forms are known to one of skillin the art.

The formulations to be used for in vivo administration must be sterile.The compositions of the invention can be sterilized by conventional,well known sterilization techniques. For example, sterilization isaccomplished readily by filtration through sterile filtration membranes.The resulting solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile solution prior to administration. The process offreeze-drying is often employed to stabilize polypeptides for long-termstorage, particularly when the polypeptide is relatively unstable inliquid compositions. A lyophilization cycle is usually composed of threesteps: freezing, primary drying, and secondary drying; Williams andPolli, Journal of Parenteral Science and Technology, Volume 38, Number2, pages 48-59 (1984). In the freezing step, the solution is cooleduntil it is adequately frozen. Bulk water in the solution forms ice atthis stage. The ice sublimes in the primary drying stage, which isconducted by reducing chamber pressure below the vapor pressure of theice, using a vacuum. Finally, sorbed or bound water is removed at thesecondary drying stage under reduced chamber pressure and an elevatedshelf temperature. The process produces a material known as alyophilized cake. Thereafter the cake can be reconstituted prior to use.

The standard reconstitution practice for lyophilized material is to addback a volume of pure water (typically equivalent to the volume removedduring lyophilization), although dilute solutions of antibacterialagents are sometimes used in the production of pharmaceuticals forparenteral administration; Chen, Drug Development and IndustrialPharmacy, Volume 18, Numbers 11 and 12, pages 1311-1354 (1992).

Excipients have been noted in some cases to act as stabilizers forfreeze-dried products; Carpenter et al., Developments in BiologicalStandardization, Volume 74, pages 225-239 (1991). For example, knownexcipients include sugar alcohols (including mannitol, sorbitol andglycerol); sugars (including glucose and sucrose); and amino acids(including alanine, glycine and glutamic acid).

In addition, sugar alcohols and sugars are often used to protectpolypeptides from freezing and drying-induced damage and to enhance thestability during storage in the dried state. In general, sugars, inparticular disaccharides are effective in both the freeze-drying processand during storage. Other classes of molecules, including mono- anddi-saccharides and polymers such as PVP, also have been reported asstabilizers of lyophilized products.

For injection, the pharmaceutical formulation and/or medicament can be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze-dried, rotary-dried or spray-dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they can denature or aggregate as a resultof exposure to moisture at 3TC, resulting in a loss of biologicalactivity and possible changes in immunogenicity or other functionalproperties. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

The formulations of the invention can be designed to be short-acting,fast-releasing, long-acting, or sustained-releasing as described herein.Thus, the pharmaceutical formulations also can be formulated forcontrolled release or for slow release.

Specific dosages can be adjusted depending on conditions of disease,age, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

The specific binding agent or antibody is administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions include intravenous,intraarterial, intraperitoneal, intramuscular, intradermal orsubcutaneous administration. In addition, the specific binding agent orantibody is suitably administered by pulse infusion, particularly withdeclining doses of the specific binding agent or antibody. Preferably,the dosing is given by injections, most preferably intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic. Other administration methods are contemplated,including topical, particularly transdermal, transmucosal, rectal, oralor local administration e.g. through a catheter placed close to thedesired site. Most preferably, the antibody is administeredintravenously in a physiological solution at a dose ranging between 0.01mg/kg to 100 mg/kg at a frequency ranging from daily to weekly tomonthly (e.g. every day, every other day, every third day, or 2, 3, 4,5, or 6 times per week), preferably a dose ranging from 0.1 to 45 mg/kg,0.1 to 15 mg/kg or 0.1 to 10 mg/kg at a frequency of 2 or 3 times perweek, or up to 45 mg/kg once a month.

Administration to Brain

A variety of approaches are known in the art to effect administration ofcompounds to the brain. For example, a compound can be administered bydirect intraventricular or intrathecal injection, preferably via slowinfusion to minimize impact on brain parenchyma. The desired drug alsocan be delivered using a slow release implant in the brain, or (wherethe drug is a polypeptide) implanted recombinant cells that produce thedrug. The blood brain barrier (BBB) can be permeabilized concomitantwith drug administration, to permit movement of the drug across the BBB.Permeablizing agents include osmotic agents, such as hypertonicmannitol, or another permeabilizing agent such as bradykinin, analkylglycerol, ultrasound, electromagnetic radiation or parasympatheticinnervation.

Alternatively, receptor-mediated transport can be utilized to administerdrug to the brain. It is known in the art that peptides and proteinsthat directly cross the BBB may serve as carriers for selectivetherapeutic agents that allow the therapeutic agents to cross the BBBafter delivery into the bloodstream (Pan et al., Brain Research Reviews,46:32-43, 2004; Misra et al., J. Pharm. Pharmaceut. Sci., 6:252-273,2003; Begley, Pharmacol Ther. 2004 October; 104(1):29-45; Poduslo, USApp. Pub. No. 2003/0082191; Poduslo et al., Biochem., 43:6064-6075,2004). For example, Poduslo, WO 03/020212 describes conjugation ofantibodies to amyloid-beta protein fragments which are then taken up bylow-density lipoprotein receptor related protein-1, a transporter at theBBB. Other examples of peptides which cross the BBB include transferrinwhich binds to the transferrin receptor, a transporter at the BBB;monoclonal antibodies to the transferrin receptor such as OX26;cell-penetrating peptides such as TAT transduction domain, penetratin,or Syn BI; and RAP which binds to low-density lipoprotein receptorrelated protein-2, another transporter at the BBB (see Pan et al., JCell Sci. 2004 Oct. 1; 117(Pt 21):5071-8).

Receptor-mediated drug delivery to the brain can employ chimeric peptidetechnology, wherein a non-transportable drug is conjugated to a BBBtransport vector. The latter can be a modified protein orreceptor-specific monoclonal antibody that undergoes receptor-mediatedtranscytosis through the BBB in vivo. Conjugation of drug to transportvector is facilitated with chemical linkers, avidin-biotin technology,polyethylene glycol linkers, or liposomes. Multiple classes oftherapeutics have been delivered to the brain with the chimeric peptidetechnology, including peptide-based pharmaceuticals, anti-sensetherapeutics including peptide nucleic acids (PNAs), and small moleculesincorporated within liposomes. Alternatively, the drug can beencapsulated in a liposome or nanoparticle which is then linked to theBBB transport vector.

Administration with Other Agents

The antibodies can be concurrently administered with otheranti-amyloidgenic therapeutic agents. Concurrent administration includesadministration of the two different therapeutic agents at differenttimes and at different routes, as long as there is some overlap in thetime during which the agents are exerting their therapeutic effects.

Exemplary anti-amyloidgenic agents known in the art include otheranti-amyloid-beta antibodies, anti-inflammatories known in the art(e.g., NSAIDs and Cox-2 inhibitors) that reduce the pathogenic effectsof amyloid accumulation, cholesterol lowering drugs, β-secretaseinhibitors, or anti-inflammatories that reduce the inflammatory responsedue to the administration of Aβ antibody or that allow monitoring of theside effects of the anti-Aβ antibody.

Administration of medicaments according to the present invention can beachieved via any common route. Although the intravenous route is apreferred embodiment, other routes of administration are contemplated.This includes oral, nasal, buccal, rectal, vaginal or topical.Alternatively, administration may be by orthotopic, intradermal,subcutaneous, intramuscular or intraperitoneal.

In another preferred embodiment, the medicament according to the presentinvention comprising a polypeptide according to the invention isformulated for a combined administration with a second medicament forthe respective disease. Examples for such therapies would be inhibitorsof acetylcholine esterase or NMDA (N-methyl-D-aspartate) receptorantagonists. The administration of the medicament according to theinvention can be prior to, simultaneously with or after administrationof the second medicament.

In a further aspect the present invention relates to a method oftreatment and/or prevention of Alzheimer's disease, Down's syndrome,Dementia with Lewy bodies, fronto-temporal dementia, cerebral amyloidangiopathy or amyloidoses comprising administering one or more of theabove mentioned polypeptides of the invention to a subject in needthereof.

The present invention also relates to the use of a polypeptidecomprising an amino acid sequence of an Aβ polypeptide, wherein thesequence of the AD polypeptide has at least the sequence according toSEQ ID NO:2, i.e. Aβ(21-37) and at most the sequence according to SEQ IDNO:4, i.e. Aβ(12-40) for isolation and separation of a polypeptideaccording to the present invention from a sample.

Detecting and Measuring the Progression of Disease Peptides

In another aspect, Aβ peptides are provided that are useful fordetecting and/or measuring the progression of Alzheimer's and otherneurodementing diseases.

The inventors identified two Aβ epitope sequences recognized by Aβautoantibodies isolated from serum of AD patients as well as of healthycontrol individuals. The Aβ autoantibodies of healthy controlindividuals were found to specifically recognize the C-terminal part ofthe Aβ sequence, namely Aβ(21-37) (SEQ ID NO: 2) or any other sequencecomprising Aβ(21-37). Furthermore Aβ patients have an increased fractionof antibodies recognizing the N-terminus, in particular the Aβ(4-10)epitope of the Aβ polypeptide (SEQ ID NO: 3), while having a decreasedfraction of the antibodies recognizing the C-Terminus, in particular theAβ(21-37) epitope or any other sequence comprising Aβ(21-37) of the Aβpolypeptide. This provides the basis for a new, early Aβ diagnosticmethod by determination of AD antibodies, wherein an elevated level ofAβ autoantibody (Aβ21-37) is a positive indicator, i.e. “healthy”, whilean elevated level of “plaque specific” Aβ(4-10)-antibody is a negativeindicator, i.e. “sick”, with respect to the prognosis for diseaseprogression in a subject.

In one aspect, the present invention relates therefore to a polypeptidecomprising an amino acid sequence of an Aβ peptide, wherein the Aβpeptide has at least the sequence according to SEQ ID NO: 2 and at mostthe sequence according to SEQ ID NO: 4.

A polypeptide according to the invention having a sequence of Aβ as setforth above may be for instance the Aβ(21-37) fragment of amyloid betaitself. Other possible embodiments could comprise for example Aβ(20-37),Aβ(12-37), Aβ(12-40) (SEQ ID NO: 4), Aβ(20-40), Aβ(21-40) and so forth.This polypeptide fragment can be joined to other moieties. This meansthat a polypeptide according to the invention can comprise besides theAβ(21-37) portion other polypeptide sequences or non-polypeptidestructures or portions. The moieties attached to the Aβ polypeptidefragments may facilitate the performance of the methods according to thepresent invention. In other aspects, Aβ polypeptides can form oligomers.Examples of such oligomers include, but are not limited to, oligomericforms of Aβ(1-40) or oligomeric forms of Aβ(12-20) or oligomeric formsof Aβ(21-37).

Thus, in a preferred embodiment the polypeptide according to theinvention additionally comprises other moieties such as tags or markers,which facilitate in particular the attachment of the polypeptide to acarrier. In particular such tags can provide for the immobilisation ofthe polypeptide on a carrier coated with the respective antagonist.Examples for such moieties are biotin, streptavidin, GST, HIS,STREP-tag, Myc, HA, poly-L-lysine, poly-L-lysine-L-alanine copolymers,poly-Aib (alpha-aminoisobutyric acid), poly-β-alanine, poly-L-alanine,poly-D-lysine, poly-D-lysine-D-alanine copolymers, poly-D-alanine, orcombinations of poly-L- and -D-amino acids. The polypeptide comprising asequence according to SEQ ID NO 3 and at most the sequence according toSEQ ID NO: 5, as used in some of the methods of the invention, can alsocomprise such additional moieties.

The above mentioned peptide markers can be directly fused to thepolypeptides having e.g. the sequence of SEQ ID NO 2 or 3, respectively.If a higher flexibility between the marker/tag and the e.g. Aβ(21-37)peptide is desired, linkers can be introduced. Such linkers can be forinstance polyglycine or -alanine linkers. Biotin, a non-peptidicsubstance, can be covalently linked to a polypeptide of the presentinvention. A multitude of possible combinations of markers and carriersfor the immobilization of a polypeptide of the present invention isknown from the prior art.

In a preferred embodiment the region of the above mentioned polypeptidehaving e.g. the sequence according to SEQ ID NO: 2 is not in β-sheetconformation. The β-pleated sheet conformation of Aβ has been shown tobe responsible for neurotoxicity. Thus, Aβ(21-37) antibodies in ahealthy individual recognize in particular the Aβ(21-37) region or anyother sequence comprising Aβ(21-37), if it is in random coil oralpha-helix conformation. In another preferred embodiment, therefore,the region of the above mentioned polypeptide having the sequenceaccording to SEQ ID NO: 2 or any other sequence comprising Aβ(21-37) isflanked by amino acid sequences, which prevent or reduce β-sheetformation of the polypeptide region having the sequence according to SEQID NO: 2. Preferably said flanking amino acid sequences are located inclose proximity to the N- and/or C-terminal ends of the Aβ sequencestretch, e.g. having the sequence according to SEQ ID NO: 2 and/or whichflanking amino acid sequences are composed of oligomeric peptidescomprising, for example, L-alanine, D-alanine, Aib(alpha-aminoisobutyric acid), β-alanine, D-valine, L-glycine, D-glycineand/or related hydrophobic amino acids. Particularly preferred asflanking amino acid sequences are oligomeric peptides such as-(L-alanine)n-, -(D-Alanine)n-, -(Aib)n-, -(β-alanine)n-, -(D-valine)n-,-(L-glycine)n-, -(D-glycine)n- wherein n ranges preferably from about 2to about 6. Such flanking regions also ensure, that the epitopeAβ(21-37) of the polypeptides of the invention is not present in β-sheetconformation and thus accessible to the Aβ(21-37) autoantibodies in thesamples. In some embodiments, the β-amyloid polypeptide is in oligomericform.

However, in a particular embodiment, the polypeptide according to thepresent invention is a minimal polypeptide that has no further sequencesthan the sequence of Aβ(20-37), Aβ(12-37), Aβ(12-40), Aβ(20-40) orAβ(21-40). In particular oligomeric forms of said polypeptides arepreferred embodiments of the invention.

However, it has to be understood, that other polypeptides than the onesmentioned above, which bind specifically to a first monoclonal antibody,which first antibody is capable of binding specifically to a sequence asdenoted in SEQ ID NO 2 or any other sequence comprising Aβ(21-37), butwhich polypeptides do not bind specifically to a second monoclonalantibody, which second monoclonal antibody is capable of bindingspecifically to a sequence as denoted in SEQ ID NO 3, can also be usedfor practicing the methods of the present invention.

In a preferred embodiment the polypeptide according to the invention isdirectly synthesized by conventional polypeptide synthesis methods (seealso Example 5). Similar approaches are suitable for generation of apolypeptide comprising an amino acid sequence of an Aβ peptide, whereinthe Aβ peptide has at least the sequence according to SEQ ID NO: 3 andat most the sequence according to SEQ ID NO: 5.

Alternatively, the polypeptide according to the invention can also beobtained by in vitro translation or via a recombinant expression system.In order to express a polypeptide according to the invention arespective nucleic acid expression construct has to be generated. Aperson skilled in the art will clearly see several ways to constructappropriate expression system harboring a nucleic acid sequence encodingfor a polypeptide according to the invention. The construct issubsequently expressed in a suitable host cell and the polypeptide isisolated. For this purification step the above mentioned markers and/ortags can be used as well.

The inventors found in serum of AD patients an additional fraction ofantibodies directed against a N-terminal epitope of AD peptide, which isnot present, or only in low abundance, in healthy individuals. Thus, thedetection of such antibodies is indicative for the diagnosis, stageand/or progression of AD. To distinguish between Aβ(21-37) on one handand Aβ(4-10) or other antibodies directed against the N-terminal part ofAβ, for such diagnosis methods and assays a polypeptide can be usedcomprising an amino acid sequence of an Aβ peptide, wherein the Aβpeptide has at least the sequence according to SEQ ID NO: 3 (Aβ(4-10))and at most the sequence according to SEQ ID NO: 5 (Aβ(1-20)).

In one embodiment, the invention relates to a polypeptide comprising anamino acid sequence of an Aβ peptide, wherein the Aβ peptide has atleast the sequence according to SEQ ID NO: 2 and at most the sequenceaccording to SEQ ID NO: 4.

The polypeptide according to the invention thus comprises at least asequence stretch identical to the AD peptide amino acid sequence rangingfrom amino acid 21 to amino acid 37 of SEQ ID NO: 1. This peptidesequence is denoted herein as SEQ ID NO: 2 or Aβ(21-37), respectively. Apolypeptide according to the present invention can exhibit also longersequence stretches of the Aβ peptide sequence, going beyond theAβ(21-37) sequence. However, the length of the Aβ peptide sequencestretch comprised by the polypeptide according to the invention shouldnot range further than from amino acid 12 to amino acid 40 of the ADpeptide, also denoted herein as SEQ ID NO:4 or Aβ(12-40). This ensures,that the Aβ sequence stretch of the polypeptide of the invention doesnot comprise amino acids relevant for the binding of the plaque specificAβ antibody found in patients with AD. The polypeptide according to thepresent invention can also comprise other amino acid sequences. Forexample, tags, markers, binding domains, activation domains or similarfunctional moieties can be fused to the Aβ sequence stretch, forinstance, to provide for a better binding of the polypeptide accordingto the invention to certain surfaces. Analogous, the polypeptideaccording to the present invention can be modified for certain purposes,such as covalent coupling to a fluorophor or chromophor, biotin, or thelike. In certain cases the non-Aβ sequence stretches of the polypeptideaccording to the invention provide for a structural stabilisation of thea Aβ(21-37) sequence stretch, preventing or reducing the formation ofβ-sheet conformation in this region.

In another embodiment, the inventive polypeptides bind specifically tooligomeric forms of β-amyloid polypeptide. By way of non-limitingexample, the polypeptides can bind oligomeric forms of Aβ(1-40) oroligomeric forms of Aβ(12-40) or oligomeric forms of Aβ(21-37). In oneaspect, the inventive polypeptides are capable of binding specificallyto oligomeric forms of Aβ(1-40) when incubated overnight with stirringin 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 at 4° C.

In a preferred embodiment the above mentioned polypeptide additionallycomprises one or more moieties such as tags or markers, in particularbiotin, streptavidin, GST, HIS, STREP-tag, Myc, HA, poly-L-Iysine,poly-L-lysine-L-alanine copolymers, poly-Aib (alpha-aminoisobutyricacid), poly-β-alanine, poly-L-alanine, poly-D-lysine,poly-D-lysine-D-alanine copolymers, poly-D-alanine, or combinations ofpoly-L- and -D-amino acids.

In a preferred embodiment the region of the above mentioned polypeptide,which has the sequence according to SEQ ID NO: 2 or any other sequencecomprising Aβ(21-37) is not in a β-sheet conformation. In an even morepreferred embodiment this region is in random coil or exhibits α-helixconformation.

In another preferred embodiment the region of the above mentionedpolypeptide having e.g. the sequence according to SEQ ID NO: 2 or anyother sequence comprising Aβ(21-37) is flanked by amino acid sequences,which prevent or reduce β-sheet formation of the Aβ polypeptide regionhaving e.g. the sequence according to SEQ ID NO: 2 or any other sequencecomprising Aβ(21-37), in particular wherein said flanking amino acidsequences are located in close proximity to the N- and/or C-terminalends of the Aβ sequence and which flanking amino acid sequences arecomposed of oligomeric peptides comprising, for example, L-alanine,D-alanine, Aib (alpha-aminoisobutyric acid), β-alanine, D-valine,L-glycine, D-glycine and/or related hydrophobic amino acids.Particularly preferred as flanking amino acid sequences are oligomericpeptides such as -(L-alanine)_(n)-, -(D-Alanine)_(n)-, -(Aib)_(n)-,-(β-alanine)_(n)-, -(D-valine)_(n)-, -(L-glycine)_(n)-,-(D-glycine)_(n)- wherein n ranges preferably from about 2 to about 6.In some embodiments, the β-amyloid polypeptide is in oligomeric form.

Furthermore, the present invention relates to the use of a polypeptideaccording to the present invention and/or of a polypeptide comprising anamino acid sequence of an Aβ peptide, wherein the Aβ peptide has atleast the sequence according to SEQ ID NO: 3 and at most the sequenceaccording to SEQ ID NO: 5, for diagnostic assays. Methods for suchdiagnostic assays are exemplified below.

Methods

In another aspect the present invention relates to a method fordiagnosing a neurodementing disease, the method comprising the followingsteps:

-   -   a) incubating a polypeptide according to the present invention        immobilized on a carrier with a sample derived from a subject,        subsequently    -   b) separating said sample from the carrier, and    -   c) detecting polypeptides bound to the immobilized polypeptide        of step a).

The present invention also relates to a method for diagnosing aneurodementing disease, the method comprising the following steps:

-   -   a) incubating a polypeptide according to the present invention        with a sample derived from a subject, and subsequently    -   b) incubating the sample with a carrier having a binding        affinity for the polypeptide according to the present invention,    -   c) separating said sample from the carrier, and    -   d) detecting polypeptides bound to the polypeptide according to        the present invention, said polypeptide being bound to the        carrier.

In another aspect the present invention relates to methods of diagnosisof a neurodementing disease, which utilize a polypeptide or proteinwhich comprises at least a sequence stretch identical to the Aβ peptideamino acid sequence ranging from amino acid 4 to amino acid 10 of SEQ IDNO: 1. This peptide sequence is denoted herein as SEQ ID NO: 3 orAβ(4-10) or the N-terminal peptide, respectively. Such a polypeptide canexhibit also longer sequence stretches of the Aβ peptide sequence, goingbeyond the Aβ(4-10) sequence. However, the length of the Aβ peptidesequence stretch comprised by such a polypeptide should preferably notrange further than from amino acid 1 to amino acid 20 of the Aβ peptide,also denoted herein as SEQ ID NO:5 or Aβ (1-20). This ensures, that theAβ sequence stretch of the polypeptide of the invention does notcomprise amino acids relevant for the binding of the Aβ autoantibodyfound in healthy individuals and directed to Aβ(21-37). The N-terminalpolypeptide can comprise besides the Aβ sequence stretch other aminoacid sequences, moieties and modifications as well, as already mentionedfor the polypeptide according to the invention.

Thus, the present invention also relates to a method for diagnosing aneurodementing disease, the method comprising the following steps:

-   -   a) incubating a polypeptide immobilized on a carrier with a        sample derived from a subject, wherein the polypeptide comprises        an amino acid sequence of an Aβ peptide, wherein the AD peptide        has at least the sequence according to SEQ ID NO: 3 and at most        the sequence according to SEQ ID NO: 5, subsequently    -   b) separating said sample from the carrier, and    -   c) detecting polypeptides bound to the immobilized polypeptide        of step a).

The present invention also relates to a method for diagnosing aneurodementing disease, the method comprising the following steps:

-   -   a) incubating a polypeptide with a sample derived from a        subject, wherein the polypeptide comprises an amino acid        sequence of an Aβ peptide, wherein the Aβ peptide has at least        the sequence according to SEQ ID NO: 3 and at most the sequence        according to SEQ ID NO: 5, and subsequently    -   b) incubating the sample with a carrier having a binding        affinity for the polypeptide comprising an amino acid sequence        of an Aβ peptide, wherein the Aβ peptide has at least the        sequence according to SEQ ID NO: 3 and at most the sequence        according to SEQ ID NO: 5,    -   c) separating said sample from the carrier, and    -   d) detecting polypeptides bound to the polypeptide, which        comprises an amino acid sequence of an Aβ peptide, wherein the        AD peptide has at least the sequence according to SEQ ID NO: 3        and at most the sequence according to SEQ ID NO: 5, said        polypeptide being bound to the carrier.

The present invention also relates to a method for diagnosing aneurodementing disease, the method comprising the following steps:

-   -   a) incubating a polypeptide according to the present invention        immobilized on a carrier with a cell containing sample derived        from a subject,    -   b) separating said sample from the carrier, and    -   c) detecting cells bound to the immobilized polypeptide of step        a).

The present invention also relates to a method for diagnosing aneurodementing disease, the method comprising the following steps:

-   -   a) incubating a polypeptide according to the present invention        with a cell containing sample derived from a subject, and        subsequently    -   b) incubating the sample with a carrier having a binding        affinity for a polypeptide of the present invention,    -   c) separating said sample from the carrier, and    -   d) detecting cells bound to the polypeptide according to the        present invention, said polypeptide being bound to the carrier.

The present invention also relates to a method for diagnosing aneurodementing disease, the method comprising the following steps:

-   -   a) incubating a polypeptide immobilized on a carrier with a cell        containing sample derived from a subject, wherein the        polypeptide comprises an amino acid sequence of an Aβ peptide,        wherein the Aβ peptide has at least the sequence according to        SEQ ID NO: 3 and at most the sequence according to SEQ ID NO: 5,    -   b) separating said sample from the carrier, and    -   c) detecting cells bound to the immobilized polypeptide of step        a).

The present invention also relates to a method for diagnosing aneurodementing disease, the method comprising the following steps:

-   -   a) incubating a polypeptide with a cell containing sample        derived from a subject, wherein the polypeptide comprises an        amino acid sequence of an Aβ peptide, wherein the Aβ peptide has        at least the sequence according to SEQ ID NO: 3 and at most the        sequence according to SEQ ID NO: 5, and subsequently    -   b) incubating the sample with a carrier having a binding        affinity for the polypeptide comprising an amino acid sequence        of an Aβ peptide, wherein the Aβ peptide has at least the        sequence according to SEQ ID NO: 3 and at most the sequence        according to SEQ ID NO: 5,    -   c) separating said sample from the carrier, and    -   d) detecting cells bound to the polypeptide, which comprises an        amino acid sequence of an Aβ peptide, wherein the AD peptide has        at least the sequence according to SEQ ID NO: 3 and at most the        sequence according to SEQ ID NO: 5, said polypeptide being bound        to the carrier.

In a further embodiment of the invention the polypeptide, whichcomprises an amino acid sequence of an Aβ peptide, wherein the Aβpeptide has the sequence according to SEQ ID NO: 3 and at most thesequence according to SEQ ID NO: 5, can comprise, as mentioned above,additional moieties such as tags or markers, in particular biotin,streptavidin, GST, HIS, STREP-tag, Myc, HA, poly-L-lysine,poly-L-lysine-L-alanine copolymers, poly-Aib (alpha-aminoisobutyricacid), poly-β-alanine, poly-L-alanine, poly-D-lysine,poly-D-lysine-D-alanine copolymers, poly-D-alanine, or combinations ofpoly-L- and -D-amino acids. In a preferred embodiment, a polypeptide ofthe invention is identical to the polypeptide, which comprises an aminoacid sequence of an Aβ peptide, wherein the Aβ peptide has the sequenceaccording to SEQ ID NO: 3 and at most the sequence according to SEQ IDNO: 5, except for the sequence stretch covering the Aβ-sequence.

In a preferred embodiment of the methods of the invention employing apolypeptide according to the invention a solvent is present in theincubation step(s), which prevents or reduces β-sheet formation of thepolypeptide region having e.g. the sequence according to SEQ ID NO: 2.Preferred solvents may be trifluoroethanol (TFE), hexafluoro-isopropanolor similar solvents to stabilize peptide conformations and to prevent orreduce β-sheet formation. Preferably, the solvent is present in anaqueous solution comprising for example 5-10 mM phosphate buffer and 150mM NaCl. In an even more preferred embodiment the concentration of TFE,hexafluoro-isopropanol and the like in the aqueous solution ranges fromabout 1 to about 5%, preferably from about 1% to about 2%.

In a preferred embodiment the methods of the invention comprise anadditional step, wherein at least one washing step is performed beforethe detecting step.

In a preferred embodiment of the methods according to the presentinvention, relating to the detection of cells, the methods are carriedout in form of affinity chromatography, in particular immunoaffinitychromatography.

The amount of polypeptides, e.g. antibodies, binding to a polypeptideaccording to the present invention, e.g. Aβ (21-37) or any othersequence comprising Aβ (21-37), in a sample of a subject is an indicatorfor the status of the subject with regard to the development and/orprogression of AD. The higher the concentration of polypeptides directedagainst the Aβ (21-37) sequence stretch, the higher the protectivecapacity and the lower the risk of development and/or progression of AD.The amount of polypeptides, e.g. antibodies, directed against theAβ(4-10) epitope, in a sample of a subject, is an indicator for thestatus of the subject with regard to the development and/or progressionof AD as well. However, in this case the situation is vice versa. Thehigher the concentration of polypeptides directed against the Aβ (4-10)sequence stretch, the higher the risk of development and/or progressionof AD. For diagnostic reasons, the methods of detection according to thepresent invention can thus be performed individually, but also incombination in order to provide for a more specific diagnosis.

Thus, in a further embodiment of the invention a method according to theinvention employing a polypeptide according to the invention is carriedout in combination with a method of the present invention employing apolypeptide comprising an amino acid sequence of an Aβ peptide, whereinthe Aβ peptide has at least the sequence according to SEQ ID NO: 3 andat most the sequence according to SEQ ID NO: 5. The method employing apolypeptide of the present invention can be performed simultaneously,prior to or after the second method.

It is also possible to diagnose the neurodementing disease by way of anindirect approach. A method according to the present invention—utilizingeither a polypeptide according to the present invention or a polypeptidecomprising an amino acid sequence of an Aβ peptide, wherein the Aβpeptide has at least the sequence according to SEQ ID NO: 3 and at mostthe sequence according to SEQ ID NO: 5—is combined with a similarmethod, which only differs from the methods of the present invention byutilizing polypeptide comprising a polypeptide having the length ofAβ(1-40) or Aβ(1-42), i.e. full length Aβ peptide instead of the shorterAβ sequence stretches utilized by the methods of the present invention.In such a scenario, the two methods are carried out independently fromeach other and the results of the methods are compared. To illustratethis concept, the following example is given:

-   -   1) The method utilizing the Aβ full length polypeptide sequence        yields the amount of all polypeptides (or cells producing such        polypeptides, respectively) in the sample directed against full        length Aβ (Result A).    -   2) On the other hand a method of the present invention utilizing        a polypeptide of the present invention yields the amount of all        polypeptides (or cells producing such polypeptides,        respectively) in the sample directed against a polypeptide of        the present invention such as Aβ(21-37) or any other sequence        comprising Aβ(21-37 (Result B).    -   3) A person skilled in the art can now easily deduce the amount        of polypeptides in the sample directed against an epitope at the        N-terminus of Aβ, in particular against epitope Aβ(4-10), by        subtracting Result B from Result A.

Analogously, the amount of polypeptides (or cells producing suchpolypeptides, respectively) in the sample directed against a polypeptideof the present invention such as Aβ(21-37) or any other sequencecomprising Aβ(21-37) can be obtained by subtracting the results obtainedwith a polypeptide comprising an amino acid sequence of an Aβ peptide,wherein the Aβ peptide has at least the sequence according to SEQ ID NO:3 and at most the sequence according to SEQ ID NO: 5, e.g. Aβ(4-10),from the results obtained with Aβ full length.

Thus, the present invention also refers to a method for diagnosing aneurodementing disease, wherein the methods according to the presentinvention comprise the following steps:

-   -   i) performing a first method according to the present invention        as set forth above,    -   ii) performing a second method according to the present        invention proviso that the polypeptide to be incubated in        step a) of said second method comprises the full length amino        acid sequence of Aβ peptide, and    -   iii) comparing the result obtained from step i) with the result        of step ii).

A person skilled in the art will understand that it does not matter forthe above method whether step i) is carried out prior to, simultaneouslywith or after step ii).

In one embodiment the polypeptides to be detected in the methods of theinvention are antibodies, in particular an Aβ(21-37) autoantibody or anAβ(4-10) autoantibody.

In a preferred embodiment the methods according to the invention arecarried out for diagnosing Alzheimer's disease, Down's syndrome,Dementia with Lewy bodies, fronto-temporal dementia, cerebral amyloidangiopathy, and/or amyloidoses.

In a further aspect the present inventions relates to a carriercomprising a polypeptide according to the invention. In a preferredembodiment the carrier additionally comprises a second polypeptidecomprising an amino acid sequence of an Aβ peptide, wherein the Aβpeptide of the second polypeptide has at least the sequence according toSEQ ID NO: 3 and at most the sequence according to SEQ ID NO: 5.

The carriers according to the invention and used in the methods of theinvention can be of any suitable material capable of bindingpolypeptides such as beads, in particular magnetic beads or sepharosebeads, membranes, in particular polyvinylidene fluoride ornitrocellulose membranes, glass, sepharose matrices, gold surfaces,synthetic surfaces, in particular microtiter plates. For certainembodiments, the surface of the carriers can be coated with agents,which are, for instance, capable of binding to the tags and markersmentioned above.

Detection

A variety of assays can be employed in the inventive methods to detector measure antibody titer against the Aβ peptides of interest. Exemplaryassays include, but are not limited to, ELISA, ELISPOT, Western-Blot,Dot Blot, Protein-Chip, surface plasmon resonance assay,immunoprecipitation or co-immunoprecipitation, or affinitychromatography, in particular immunoaffinity chromatography.

In a preferred embodiment, the methods according to the inventioncomprising the detection of polypeptides in step c) or d), respectively,represent an ELISA. In this case the carrier is for example a microtiterplate. The separation of sample and carrier is achieved by removing thesample liquid from the microtiter plate and/or by washing the microtiterplate after the incubation. Preferably, the bound polypeptide is in thisscenario an antibody and this antibody can be detected for example via asecondary antibody, coupled with e.g. alkaline phosphatase (AP), and theaddition of a substrate for AP resulting in the turn over of thesubstrate into, for example, a colored compound detectable by an opticaldevice. A person skilled in the art and familiar with ELISA techniqueswill know several variations of the ELISA concept, which can be appliedto the methods of the present invention as well.

Alternatively, in another preferred embodiment, the methods according tothe invention comprising the detection of polypeptides in step c) or d),respectively, represent an ELISPOT assay. In this case the carrier isfor example a nitrocellulose plate. The incubation step of the carrierwith the sample provides in this scenario enough time for a cell in thesample to produce sufficient amounts of antibody. The separation ofsample and carrier is achieved by removing the sample liquid from thenitrocellulose plate and/or by washing the nitrocellulose plate afterthe incubation. Preferably, the bound polypeptide is an antibody andthis antibody can be detected, for example via a secondary antibody,coupled with e.g. alkaline phosphatase (AP), and the addition of asubstrate for AP, such as BCIP/NBT (Bromo-chloro-indoryl phosphate/NitroBlue Tetrazolium) resulting in the turn over of the substrate into, forexample, a deep purple stain detectable visually or by an opticaldevice. A person skilled in the art and familiar with ELISPOT techniqueswill know several variations of the ELISPOT concept, which can beapplied to the methods of the present invention as well.

In a further embodiment, the methods according to the inventioncomprising the detection of polypeptides in step c) or d), respectively,represent a Western Blot or Dot Blot assay. In this case the carrier isfor example a nitrocellulose membrane. For the Western Blot, on thenitrocellulose membrane is either immobilized a polypeptide as used instep a) of the methods according to the present invention or a substancewith binding affinity for a polypeptide as used in step a) of themethods according to the present invention. The nitrocellulose orsimilar membrane (PVDF etc.) itself can provide for the binding affinityto the polypeptide as used in step a) of the methods according to thepresent invention. The separation of sample and carrier is achieved byremoving the sample liquid from the nitrocellulose membrane and/or bywashing the nitrocellulose membrane after the incubation. Preferably,the bound polypeptide is an antibody and this antibody is for exampledetected via a labeled secondary antibody, e.g. coupled with horseradishperoxidase, and subsequent luminescent reaction and detection. A personskilled in the art and familiar with Western Blot/Dot blot techniqueswill know several variations of the Western Blot/Dot blot concept, whichcan be applied to the methods of the present invention as well.

In a further embodiment, the methods according to the inventioncomprising the detection of polypeptides in step c) or d), respectively,represent a Protein chip, i.e. protein microarray. In this case thecarrier is for example a glass surface functionalized for bindingproteins. The separation of sample and carrier is achieved by removingthe sample liquid from the glass carrier and/or by washing the glasscarrier after the incubation. Preferably, the bound polypeptide is anantibody and this antibody is detected via a labelled secondaryantibody, coupled with e.g. a fluorescent dye, which can be detected byan optical device. A person skilled in the art and familiar with proteinchip techniques will know several variations of the protein chipconcept, which can be applied to the methods of the present invention aswell.

In a further embodiment, the methods according to the inventioncomprising the detection of polypeptides in step c) or d), respectively,represent a surface plasmon resonance analysis. In this case the carrieris for example a metal surface such as a gold surface. The separation ofsample and carrier is achieved by removing the sample liquid from themetal carrier and/or by washing the metal carrier after the incubation.Preferably, the bound polypeptide is an antibody and the binding of theantibody is detected via measuring the intensity of the reflected lightat a specific incident angle with an optical device. A person skilled inthe art and familiar with plasmon resonance analysis techniques willknow several variations of the surface plasmon resonance concept, whichcan be applied to the methods of the present invention as well.

In a further embodiment, the methods according to the inventioncomprising the detection of polypeptides in step c) or d), respectively,represent a pull down or immunoprecipitation experiment. In this casethe carrier may consist of sepharose beads. These sepharose beads arecoated for example with glutathione (for pull down assays) or with anantibody (immunoprecipitation assays). Separation of sample and carrieris achieved by removing the sample liquid from the sepharose beadsand/or by washing the sepharose beads after the incubation. Preferably,the bound polypeptide is an antibody and this antibody is detected via asubsequent Western Blot analysis of the precipitated protein complexes.A person skilled in the art and familiar with Pulldown/Immunoprecipitation techniques will know several variations ofthese concepts, which can be applied to the methods of the presentinvention as well. One such variation would be the application of aco-immunoprecipitation approach, wherein the carrier has only anindirect binding affinity for the polypeptide as used in step a) of themethods according to the present invention.

In a further embodiment, the methods according to the inventioncomprising the detection of polypeptides in step c) or d), respectively,are carried out in form of an affinity chromatography. In this case thecarrier is the matrix, e.g. sepharose within a conventionalchromatography column, to which is either linked a polypeptide as usedin step a) of the methods according to the present invention or asubstance with binding affinity for a polypeptide as used in step a) ofthe methods according to the present invention. The incubation of thesample with the carrier comprises the time frame the sample needs topass through the column. The separation of sample and carrier isachieved by eluting the sample liquid from the column and/or by washingthe column after the incubation. Preferably, the bound polypeptide is anantibody and this antibody is for example detected by elution of thebound antibody, for instance with a buffer having a high salt content,and subsequent detection of eluted polypeptide, for example by directoptical determination or by subsequent Western blot or similar analyses.A person skilled in the art and familiar with affinity chromatographytechniques will know several variations of the affinity chromatographyconcept, which can be applied to the methods of the present invention aswell. One such variation would be the application of an immunoaffinitychromatography approach, wherein the carrier is coated with antibodiesdirected against a polypeptide as used in step a) of the methodsaccording to the present invention.

In one embodiment, the methods according to the invention comprising thedetection of cells in step c) or d), respectively, are carried out inform of an affinity chromatography. In this case, for example, magneticbeads coated with sepharose represent the carrier, to which either apolypeptide as used in step a) of the methods according to the presentinvention is linked or a substance with binding affinity for apolypeptide as used in step a) of the methods according to the presentinvention. The separation of sample and carrier is achieved by elutingthe sample liquid from the column and/or by washing the column after theincubation. The cells bound to the matrix are for example B- or T-cells,which can be detected, after elution of the cells from the matrix, forexample by way of flow cytometry. A person skilled in the art andfamiliar with affinity chromatography and flow cytometry techniques willknow several variations of these concepts, which can be applied to themethods of the present invention as well.

Thus, the methods according to the present invention can be carried outin particularly preferred embodiments as ELISA, ELISPOT, Western-Blot,Protein-Chip, surface plasmon resonance assay, immunoprecipitation orco-immunoprecipitation, or affinity chromatography, in particularimmunoaffinity chromatography. These are all exemplifications ofdiagnostic assays. Examples for ELISA's or affinity chromatography canbe found in the examples section. In diagnostic procedures based onsurface plasmon resonance (SPR) the detection, and quantification andanalysis of binding kinetics of Aγ-epitope specific antibodies can beperformed by binding of (i) a biotinylated Aβ(21-37)- orAβ(4-10)-peptide to a avidin/streptavidin-coated SPR chip surface, or bybinding (ii) Aβ(21-37)- or Aβ(4-10)-peptides with an N-terminalThiol-group containing carboxylic acid spacer to a gold-chip surface;followed by binding and determination of the Aβ-autoantibodies. A personskilled in the art will readily know how to incorporate the methodsaccording to the present invention into one of these standard techniquesand procedures mentioned above.

It has to be understood, that although the methods according to thepresent invention can be carried out in form of one of the abovementioned detection techniques per se (ELISA, ELISPOT, Western-Blot, DotBlot, Protein-Chip, surface plasmon resonance assay,immunoprecipitation, affinity chromatography, etc.), it is also possibleto combine these detection techniques or to apply them only for thedetection step according to the invention, i.e. step c) or d),respectively, while the other steps are carried out in other formats. Itis also obvious to a person skilled in the art, that theinformation/signals obtained in the detection steps in the methods ofthe present invention can provide the basis for quantification of thisinformation/signals.

In some cases it might be of higher diagnostic value, if, instead of orin addition to the detection of polypeptides, e.g. antibodies, cellsproducing said polypeptides are detected. For example, it could be ofimportance, if in an AD patient the overall number of cells producing anAβ(21-37) autoantibody is lower than in healthy individuals, or if theamount of antibody secreted by the respective antibody producing cellsis reduced. Depending on the result this can lead to differenttherapeutic approaches. Thus, the present invention also relates to thedetection of cells producing polypeptides binding to a polypeptideaccording to the invention or binding to a polypeptide comprising anamino acid sequence of an Aβ peptide, wherein the Aβ peptide has atleast the sequence according to SEQ ID NO: 3 and at most the sequenceaccording to SEQ ID NO: 5.

As used in this invention, an immobilized polypeptide refers in thisregard to a polypeptide, which is coupled to a carrier. The coupling canbe covalently or non-covalently, it can be directly to the carrier orvia a linker/linking substance. If the immobilization occursnon-covalently, then the carrier or the linking substance exhibits aspecific binding affinity for the polypeptide according to the inventionand vice versa. Binding affinity refers to a property of a substance, inparticular a polypeptide, to associate with (an) other substance(s) andto form a stable specific dimeric or multimeric complex. Suchassociations rely usually on van der Waals- or hydrogen-bonds.

The incubating step(s) serves the purpose whereby two partners of abinding pair, i.e. having a binding affinity for each other, canassociate and four a stable complex. The temperature of the incubationstep may vary, but is usually from about 0° C. to about 40° C.,preferably from about 4 to about 37° C., even more preferred about 4°C., about 16° C., about 21° C. or about 37° C. The higher thetemperature, the shorter the time of incubation might be. For example,if the incubation temperature is 4° C. it should last for at least 12 hor over night, while 1 h is usually enough for an incubation at 37° C.If suitable, the carrier can be blocked prior to the method with asuitable blocking agent, reducing the likelihood of unspecific bindingevents. Blocking agents can be for example milk powder, BSA, fetal calfsera, or any other blocking reagent.

The detection of polypeptides bound to an immobilized polypeptide of theinvention or to a polypeptide comprising an amino acid sequence of an Aβpeptide, wherein the Aβ peptide has at least the sequence according toSEQ ID NO: 3 and at most the sequence according to SEQ ID NO: 5, can beperformed by several means. One possibility would be for example theidentification via mass spectrometrical means, for example MALDI-TOF,ESI-MS, MS-FTICR. To this purpose, immunoglobulins are first isolatedfrom, for example, a serum sample of an AD patient by protein G affinitychromatography, and subsequently Aβ-autoantibodies and Aβ-plaquespecific antibodies are e.g. isolated by Aβ-epitope-chromatography,respectively. The antibodies are then immobilized, for example, on asepharose carrier as described in the examples. The specificAβ-epitopes, Aβ(21-37) and Aβ(4-10), are then identified after bindingof full-length-Aβ-polypeptide (for example Aβ(1-40) or Aβ(1-42)),followed by proteolytic epitope-excision mass spectrometric analysisusing one or several of the proteases, trypsin, chymotrypsin, Glu-Cprotease, Asp-N-protease. After washing the affinity-bound Aβ-epitope(s)until no signal is detected in the supernatant, the specific Aβ-epitopeis eluted from the column by treatment with, typically, 0.1%trifluoroacetic acid, and identified by accurate determination of itsprotonated molecular ions; the latter molecular ion mass accuracy isentirely sufficient for identification, but can be further ascertainedby collision-induced fragmentation and tandem-MS analysis of fragmentions.

For certain embodiments, secondary antibodies labeled with a fluorescentdye or moiety (e.g. GFP) or labeled with an enzymatically activesubstance such as horseradish peroxidase, alkaline phosphatase,β-galactosidase or other related enzymes able to convert a colorlesssubstrate to a suitable dye or fluorescent product can be applied. Thedetection can also be accomplished by detecting the amount of occupiedbinding sites, i.e. utilizing a labeled Aβ(21-37) antibody, which isincubated with the carrier after the removal of the sample. The amountof bound Aβ(21-37) is in this scenario an indicator for the amount ofprior bound polypeptide. The lower the amount of subsequently boundAβ(21-37) antibody is, the more Aβ(21-37) binding polypeptides containedin the sample. The mentioned examples of detection are not to beconsidered limiting, as a person skilled in the art will readily know aplurality of methods of detection which can be used in the presentinvention.

Usually, the polypeptides bound to the immobilized polypeptide, whichare detected in step c) or d), respectively, in the methods of thepresent invention will be antibodies, in particular an Aβ(21-37)autoantibody or an Aβ(4-10) autoantibody, respectively. However, othersubstances in the human body may also bind for instance to Aβ(21-37) orAβ(4-10) polypeptide.

Likewise, the detection of cells producing a polypeptide binding to apolypeptide of the present invention or binding to a polypeptidecomprising an amino acid sequence of an Aβ peptide, wherein the Aβpeptide has at least the sequence according to SEQ ID NO: 3 and at mostthe sequence according to SEQ ID NO: 5, is accomplished by standardtechniques known to a person skilled in the art. One example would bethe analysis via flow cytometry. Another approach would be the lysis ofthe cells, DNA/RNA isolation and subsequent PCR amplification ofspecific nucleotide sequences. Besides this, in the prior art there areplurality of further possibilities published, which can be employed todetect the cells in the methods of the present invention.

In a preferred embodiment the sample or the cell containing sample,respectively, used for the methods of the present invention is derivedfrom blood, plasma, urine or cerebrospinal fluid (CSF) of a subject. Inan even more preferred embodiment the cell containing sample is derivedfrom blood and the cells are of the B-cell lineage. The sample, i.e. thesubject can be of human, rodent, bovine, porcine, canine or avianorigin. In particular the sample or the cell containing sample can bederived from human, mouse, rat, rabbit, cow, pig, dog, chicken and soforth.

A sample derived from a subject is derived from tissue or body fluid ofa subject. The subject can be a healthy individual, i.e. not sufferingfrom AD, or a “patient” suffering from a neurodementing disorder. In apreferred embodiment the sample or the cell containing sample,respectively, used for the methods of the present invention is obtainedfrom blood, plasma, urine or cerebrospinal fluid (CSF) of a subject. Inan even more preferred embodiment the cell containing sample is obtainedfrom blood and the cells are of the B-cell lineage. The sample, i.e. thesubject can be of human, rodent, bovine, porcine, canine or avianorigin. In particular the sample or the cell containing sample can bederived from human, mouse, rat, rabbit, cow, pig, dog, chicken and soforth. Possible preparation procedures of such samples are well knownfrom the prior art.

In a preferred embodiment of the methods of the present inventionemploying a polypeptide of the present invention a solvent is present inthe incubation step(s), which prevents or reduces β-sheet formation ofthe polypeptide region having the sequence according to SEQ ID NO: 2. Asmentioned above, the β-pleated sheet conformation of Aβ has been shownto be responsible for neurotoxicity. Thus, Aβ(21-37) antibodies in ahealthy individual recognize in particular the Aβ(21-37) region or anyother sequence comprising Aβ(21-37), if it is in random coil or α-helixconformation. Analogous to flanking amino acid sequences, solvents caninfluence the conformational state of the polypeptides of the invention.In particular, TFE, hexafluoro-isopropanol and so forth can be used inthe methods of the present invention, for example in the incubationstep, to prevent or reduce a β-sheet conformation of the importantepitope Aβ(21-37), thus keeping it accessible to the Aβ(21-37)autoantibodies in healthy individuals. Preferably, TFE is present in aconcentration ranging from about 1% to about 5%, preferably about 1 toabout 2%.

In a preferred embodiment the methods of the invention comprise anadditional step, wherein at least one washing step with a washingsolution is performed before the detecting step. A washing step canincrease the specificity of the later detection signal and reducesbackground signals. Preferably, the washing solution is water. Morepreferably buffers like PBS or TBS are used to ensure a constant pH. Thewashing solution can contain small amounts of detergent to increase thespecificity of the signal. If the specificity of the signal is low, thesalt concentration or the concentration of the detergent can beincreased in the washing solution.

In a further embodiment of the invention a method according to theinvention employing a polypeptide of the invention, i.e. Aβ(21-37)polypeptide, is carried out in combination with a method of the presentinvention employing a polypeptide comprising an amino acid sequence ofan Aβ peptide, wherein the Aβ peptide has at least the sequenceaccording to SEQ ID NO: 3 and at most the sequence according to SEQ IDNO: 5, i.e. Aα (4-10). The method employing a polypeptide of the presentinvention can be performed simultaneously, prior to or after the secondmethod. The comparison of the abundance of Aβ(21-37) specificpolypeptides with the abundance of Aβ(4-10) specific polypeptides willprovide for a more detailed assessment of the stage and progression ofAD.

The detecting step in the methods of the present invention provides forthe possibility to quantify the amount of polypeptides bound toAβ(21-37) or Aβ(4-10). The determined values are a measure for the stageand progression of AD. For instance, a human subject can be consideredhealthy in regard to AD, if its serum contains about 1 to 100 ng/μl ofAβ(21-37) specific polypeptides and/or about 0 ng/μl. (i.e. below thedetection limit) of the Aβ(4-10) specific polypeptides. As reference fora healthy individual might serve the average concentrations of therespective polypeptides in the serum of people in the age of 20 to 35.In contrast, a subject might suffer from a neurodementing disease or beendangered to develop a neurodementing disease, for instance, if itsserum contains about 0.01 to 5 ng/μl of Aβ(4-10) specific polypeptides,preferably about 0.05 to 1 ng/μl or even more preferably about 0.01ng/μl or if the ratio of the concentration of the plaque specificpolypeptide vs. the concentration of the Aβ(21-37) specific polypeptidesin the serum raises above 0, preferably if it is higher than 0.001,0.002, 0.003, 0.004, 0.005, 0.010, 0.015, 0.020, 0.030 or even higherthan 0.050. With aging the amount of immunoglobulin produced in a humanbody decreases naturally. Therefore, in particular cases it might benecessary to consider the age of the subject before the results obtainedwith the methods according to the present invention are evaluated. Inparticular a person about 20 to about 35 years of age might beconsidered healthy, if its, for instance, serum contains about 30 to 100ng/μl or more of Aβ(21-37) specific polypeptide, while a subject about70 to about 80 years of age can still be considered equally healthy withregard to AD with “only” 2 to 5 ng/μl of Aβ(21-37) specific polypeptidein its serum.

In a preferred embodiment the methods according to the invention arecarried out for diagnosing a neurodementing disease, Alzheimer'sdisease, Down's syndrome, Dementia with Lewy bodies, fronto-temporaldementia, cerebral amyloid angiopathy, and/or amyloidoses. All diseaseshave in common, that the concentration of Aβ-autoantibody and Aβ-plaquespecific antibody is affected by the respective disease as given abovefor AD.

In a further aspect the present invention relates to a carriercomprising a polypeptide according to the invention. In a preferredembodiment the carrier additionally comprises a second polypeptidecomprising an amino acid sequence of an Aβ peptide, wherein the Aβpeptide of the second polypeptide has at least the sequence according toSEQ ID NO: 3 and at most the sequence according to SEQ ID NO: 5.

The carriers according to the invention and used in the methods of theinvention can be of any suitable material capable of bindingpolypeptides such as beads, in particular magnetic beads or sepharosebeads, membranes, in particular polyvinylidene fluoride ornitrocellulose membranes, glass, sepharose matrices, gold surfaces,synthetic surfaces, in particular microtiter plates. For certainembodiments, the surface of the carriers can be coated with agents,which are, for instance, capable of binding to the tags and markersmentioned above. A person skilled in the art will readily know a broadvariety of different carriers and possible coatings, which can beapplied for the methods according to the invention.

Kits

In another aspect the present invention relates to a kit for thediagnosis of a neurodementing disease, wherein the kit comprises apolypeptide according to the invention. In one embodiment, a kitcomprises a second polypeptide comprising an amino acid sequence of anAβ peptide, wherein the AD peptide of the second polypeptide has atleast the sequence according to SEQ ID NO: 3 and at most the sequenceaccording to SEQ ID NO: 5. In another embodiment the kit comprises acarrier, in particular a carrier as mentioned above. In anotherembodiment, a kit comprises a first Aβ peptide comprising at least thesequence according to Aβ(30-37) and at most the sequence according toAβ(12-40), and a second Aβ peptide wherein the second Aβ peptidecomprising at least the sequence according to Aβ(4-10) and at most thesequence according to Aβ(1-20). Such kits can be used for example forroutine diagnostics in hospitals and nursing homes, for example tomonitor the progression of AD or to monitor the effectiveness of an ADtherapy.

In another aspect the present invention relates to a kit for thediagnosis of a neurodementing disease, wherein the kit comprises apolypeptide according to the invention.

In a preferred embodiment the above mentioned kit comprises a secondpolypeptide comprising an amino acid sequence of an Aβ peptide, whereinthe Aβ peptide of the second polypeptide has at least the sequenceaccording to SEQ ID NO: 3 and at most the sequence according to SEQ IDNO: 5. In an even more preferred embodiment the kit comprises a carrier,in particular a carrier as mentioned above.

The kit can include one or more containers for the AD peptides. In someembodiments, the kit contains separate containers, dividers orcompartments for the Aβ peptides and informational material. Forexample, each Aβ peptide can be contained in a bottle, vial, or syringe,and the informational material can be contained in a plastic sleeve orpacket. In other embodiments, the separate elements of the kit arecontained within a single, undivided container. For example, eachpeptide is contained in a bottle, vial, or syringe that has attachedthereto the informational material in the form of a label.

The following examples explain the invention but are not considered tobe limiting. Unless indicated differently, molecular biological standardmethods were used, as e.g., described by Sambrock and Russel, 2001,Molecular cloning: A Laboratory Manual, 3. edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

EXAMPLES Example 1 Isolation of Aβ-Antibody from an AD Patient

Immuno-isolation of the serum Aβ-antibody from an AD patient byepitope-specific affinity-chromatography was performed on aSepharose-G5Aβ(4-10) affinity matrix column. The Sepharose-G5Aβ(4-10)affinity matrix was washed with 10 ml of PBS (5 mmol L⁻¹Na₂HPO₄, 150mmol L⁻¹ NaCl, pH 7.5) and transferred into a 1.7 ml vial using 300 μlof PBS. 800 μl (1 μg/μl) of two different Aβ autoantibodies (isolatedfrom the sera of Alzheimer patients) were added and the sample wasslowly rotated overnight at 4° C. The suspension was transferred to a0.8 ml micro-column (Mobitec, Gottingen, Germany) providing thepossibility of extensive washing without significant Toss of material.The first 2 ml were collected as flow through fraction. The column waswashed with 20 ml of PBS and the last 1 ml was collected forone-dimensional electrophoresis. The affinity bound IgG was eluted with6×0.5 ml 0.1% TFA; the column was shaken gently for 15′ and the releasedantibody molecules collected in a microreaction cup. The samples werelyophilized and stored until 1D-SDS-PAGE analysis (shown in FIG. 5).

Serum samples from healthy controls from all age ranges investigatedwere also tested for the presence of plaque-antibodies (N-terminalepitope), using the Aβ(4-10) epitope column. In all investigatedsamples, non-AD control samples were devoid of detectableplaque-specific antibody.

Example 2 Isolation of Anti-Aβ(21-37)-Autoantibodies from HealthyIndividuals A. Affinity Isolation and Purification ofAnti-Aβ(21-37)-Autoantibodies

The anti-Aβ(21-37)-autoantibodies were isolated from (i), commerciallyobtainable serum immunoglobulin and (ii) from serum of healthyindividuals (HI). Isolation of antibodies was performed byAβ-epitope-specific affinity chromatography by a procedure that employeda N-cysteinyl-Aβ(12-40) column which was immobilized onUltralink-iodoacetyl-solid phase carrier as described below.

N-Cysteinyl-Aβ(12-40) (H-CVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-COOH) wassynthesized by solid phase peptide synthesis using9-fluorenylmethoxycarbonyl/t-butyl (Fmoc/tBu) chemistry on a NovaSyn TGRresin (0.23 mmole/g coupling capacity) on a semi-automated PeptideSynthesizer EPS-221 (INTAVIS, Langenfeld, Germany). The followingside-chain protected amino acid derivatives were used: Fmoc-Lys(Boc)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Trt)-OH.The synthesis was performed according to the following protocol: (i) DMFwashing; (ii) Fmoc deprotection with 2% DBU, 2% piperidine in DMF (5+10min), (iii) DMF washing, (iv) coupling of 5 equiv of Fmoc aminoacid:PyBOP:NMM in DMF (40 min), (v) coupling of 5 mol-equivalents ofFmoc amino acid:PyBOP:NMM in DMF (40 min) (vi) DMF washing (3×1 min).Due to the hydrophobic character of the C-terminal sequence of Aβ,double coupling of each amino acid was employed throughout thesynthesis. After completion of the synthesis cycles, the peptide wascleaved from the resin for 3 h using a mixture containing 95% TFA, 2.5%triisopropylsilan and 2.5% deionized water. The crude product wasprecipitated with cold tert-butylmethylether, washed three times withdiethyl ether and solubilized in 10% acetic acid (aqueous solution)prior to freeze-drying. Purification of the peptides was performed bysemipreparative HPLC; subsequent characterization by HPLC and MALDI-TOFmass spectrometric analysis ensured molecular homogeneity of thepeptide.

i) Immobilisation of CysAβ(12-40) on Ultralink Iodoacetyl Gel

Since the Aβ(12-40) sequence contains two internal lysine residues whichmight lead to side reactions in immobilization procedures using aminogroups, a specific affinity column was prepared using a cysteine residueattached to the Aβ-N-terminus, to ensure homogeneous orientation ofpeptide molecules on the column support by immobilization throughcysteinyl-S-thioether linkage. The azlactone-activated support containsan iodoacetyl group (UltraLink; Perbio, Bonn, Germany) at the end of ahexadecyl-spacer group, which was reacted with the cysteinyl-sulfhydrylgroup to yield a stable thioether linkage, in order to reduce sterichindrance and provide maximum binding capacity of the antibodies. Forcovalent attachment of the Cys-Aβ(12-40), 3.7 mg of peptide weredissolved in 50 mM Tris, 5 mM EDTA-Na coupling buffer (pH 8.5) to afinal concentration of 0.37 mg/ml. The solution was added to 1 ml ofdrained Ultralink-Iodoacetyl gel and the coupling reaction was performedfor 1 hr at 25° C. under gentle mixing, followed by 30 min reaction timewithout mixing. An aliquot of 0.5 ml of the Cys-Aβ(12-40) coupledsupport was packed into a column (2.5 ml, MoBiTec, Gottingen, Germany)allowing the solution to drain. The column was washed with 3 ml ofcoupling buffer, and non-specific binding sites on the gel were blockedfor 2×45 mM by reaction with 1 ml of 50 mM L-Cysteine.HCl in couplingbuffer. Subsequently the column was washed with 5 ml of 1 M NaCl and 5ml of 0.1 M Na-phosphate, 0.15 M NaCl (pH 7.2) and stored at 4° C. Thegel support (0.5 ml) was transferred into a 15 ml Falcon vial using 5 mlPBS and mixed with 5 ml IVIgG. After gentle shaking overnight at 4° C.,the suspension was transferred to the column using the effluent tocompletely rinse the matrix back into the column. The column was washedeight times with 10 ml of PBS followed by 2 wash cycles with 10 mlultrapure water. The affinity-bound antibodies were eluted from thecolumn with 10×0.5 ml 0.1% trifluoroacetic acid (TFA). Subsequentisolation and preparation of IgG for structural characterization andaffinity studies was performed using two different protocols:

(a) The first procedure involved adjustment to neutral pH for eachfraction collected using 0.5 M NaH₂PO₄ (pH 8) in order to maintainintegrity of the antibodies for use in affinity studies. The boundantibodies were eluted from the column with 10×0.5 ml 0.1 M glycinebuffer, pH 2.8. Each fraction was collected in a microreaction tubecontaining 35 μl 1 M Tris-HCl, pH 9. To maintain integrity of theantibodies neutral pH was adjusted immediately after elution by addingthe appropriate amount of Tris-HCl or glycine buffer. To regenerate thecolumn for further use, the column was washed once with 10 ml 10 mMsodium-phosphate buffer pH 6.8, followed by two wash cycles with 10 mlof PBS containing 1M sodium chloride and finally two wash cycles with 10ml PBS. Protein concentrations were determined by the BCA method(Pierce; Perbio, Bonn, Germany). This procedure yielded the elution ofsingle, defined antibody.(b) The bound antibodies were eluted from the column with 10×0.5 ml 0.1M glycine buffer, pH 2.8. For separation by gel electrophoresis theelution of affinity-bound antibodies was not performed by subsequent pHadjustment, in order to reduce the salt content of the samples subjectedto isoelectric focusing. Gel electrophoretic separations provided a setof defined bands of antibodies (see numbering in FIG. 12).

ii) Antibody Quantification

Antibody concentrations in the elution fractions were determined by theMicro BCA™ Protein Assay Kit method (Pierce; Perbio, Bonn, Germany). Thestock solution of 2 mg/ml of bovine albumin supplied within the MicroBCA™ Kit was used to prepare fresh standard dilutions within the range40-0.5 μg/ml. The antibodies eluted between fractions 1 to 6, withhighest concentrations in fractions 1 and 3. For quantification of eachset of 10 elution fractions, fresh albumin standard dilutions wereprepared. Results were read at 562 nm with the ELISA reader.

B. Determination of the Epitope Recognized by theAnti-Aβ(21-37)-Autoantibodies Via Epitope Excision

Autoantibodies isolated as described in Example 2A from the serum ofhealthy individuals were immobilized using a solution of 100 μgAβ(21-37) autoantibodies in 500 μl 0.2 M NaHCO₃/0.5 M NaCl (pH 8.3),which was added to n-hydroxysuccinimidyl (NHS)-activated 6-aminohexanoicacid-coupled sepharose (Sigma, St Louis, USA) and allowed to bind for 60min at 20° C. before transferring onto a microcapillary (MoBiTec,Goettingen, Germany). The column was washed five times with 6 mlblocking buffer (0.1M ethanolamine, 0.5 NaCl-pH=8.3) and between theblocking steps with 6 ml washing buffer (0.2M NaOAc, 0.5M NaCl-pH=4)each with one drop per second. Then the column was incubated for 1 h inblocking buffer, followed by another wash step: washed seven timesalternatively with 6 ml washing buffer (0.2M NaOAc, 0.5M NaCl— pH=4) andwith 6 ml blocking buffer (0.1M ethanolamine, 0.5 NaCl— pH=8.3).Finally, the column was washed with 20 ml PBS (5 mM Na2HPO4, 150 mMNaCl, pH=7.5). Then the peptides to be analyzed were applied in a molarratio of about two peptides per coupled antibody. Then the columns werewashed with 10 ml PBS wash and then 10 ml double desalted H₂O (MilliQ)to remove unspecifically bound peptides. Then elution was done byapplying 500 μl 0.1% TFA and incubating it for 15 minutes under gentleagitation. Then the TFA solution containing specifically bound peptidesis eluted and TFA solution is applied again 2 to 4 times. These eluatesare pooled and lyophilized then measured by MS.

Epitope excision was performed by application of 2-5 μg Aβ-antigen inPBS in a molar ratio of about two peptides per coupled antibody to theantibody micro column produced as described in the paragraph above for60 min at room temperature (20-25° C.). After washing, digestion wasperformed on the column for 2 h at 37° C. with 0.2 μg protease in 200 μlPBS. Unbound peptides were removed and the epitope was dissociated fromthe antibody using 500 μl 0.1% trifluoroacetic acid. After incubationfor 15 min at 20° C., this step was repeated 5 times, the epitope eluatewas lyophilized and reconstituted in 10 μl 0.1% TFA or MALDI solvent(3:2 AcCN: 0.1% TFA-better) for mass spectrometric analysis.

Epitope extraction was performed in an analogous manner, however,proteolytic digestion was performed first with the unbound antigen andthe proteolytic digest was applied directly to the antibody column. Asshown in FIG. 6, the carboxy-terminal Aβ(21-37) sequence was found to bespecifically recognized (proteolytically shielded), while N-terminalresidues of Aβ were accessible for cleavage. The extracted Epitope boundby the Aβ-autoantibody isolated from the healthy individuals exhibitedthus the amino acid sequence of Aβ(21-37). Therefore, the antibodies ofthe invention were called Aβ(21-37) autoantibodies.

FIG. 3 shows a similar experiment in which it was shown that the plaquespecific antibodies directed against Aβ(4-10) were able to shield theamino acids F4, R5, D7 from proteolytic digestion.

The specificity of the inventive antibodies was further investigatedusing Aβ(21-37) autoantibodies purified as described in Example 2A fromIVIgG. The anti-Aβ antibody ACA (based on U.S. Pat. No. 7,195,761 seeexample 5) also was evaluated. The antibodies were incubated withdifferent partial Aβ peptides as specified below and washed as describedabove in Example 2B. The elution profiles were analyzed via MS as above.

FIGS. 34 (a-d) show that antibodies of the invention specifically bindin this experimental setting to Aβ(12-40) and Aβ(20-37) but do not bindAβ(25-35), Aβ(17-28) or Aβ(31-40).

FIG. 34 (b) shows that the Aβ(21-37) autoantibodies specifically boundthe Aβ(12-40) polypeptide

FIG. 34( c) shows that Aβ(21-37) antibodies did not bind toAβ-polypeptides Aβ(25-35), Aβ(17-28) or Aβ(31-40).

FIGS. 34( d) to 34(l) show that both the immobilized ACA antibody andthe immobilized Aβ(21-37) autoantibodies bind to Aβ(1-40) and toAβ(12-40) but that only the immobilized Aβ(21-37) autoantibodiesspecifically bind to Aβ(20-37) and the ACA antibody does not. Neitherimmobilized antibody bound Aβ(17-28). In addition the immobilizedantibody ACA did not bind to Aβ(4-10).

Therefore the antibodies of the invention are unique in that they arecharacterized by specifically binding to Aβ(12-40) and Aβ(20-37),whereas they do not bind to Aβ(25-35), Aβ(17-28) or Aβ(31-40) under theexperimental conditions specified above.

The structure and conformational properties, binding affinity andspecificity of the Aβ-autoantibody epitope were further characterized byinvestigation of synthetic peptides comprising the Aβ(21-37) epitopesequence, and by fine-structure mapping using Alanine sequencemutations, H-D exchange and high resolution mass spectrometry, ELISAstudies and CD spectroscopic conformational analysis in differentsolvents. Biotinylated Aβ(21-37) peptides and peptides derivativeflanked with oligo-Glycine and -(D-Ala) spacer groups were synthesizedby solid-phase peptide synthesis according to previously describedprocedures for Aβ-peptides, and were purified by reversed-phase HPLC andcharacterized by MALDI- and ESI-mass spectrometry for molecularhomogeneity (Manea et al., 2004; Mezo et al., 2004). Comparative bindingstudies were performed with Aβ-epitope peptides comprising differentC-terminal sequence lengths, using an ELISA system (see below, 3). Theseresults established an essential function for antibody affinity of thecarboxyterminal sequence end of Aβ, comprising residues 30-37; thispartial sequence is critically involved in β-sheet formation andaggregation of AD. Thus, full binding affinity is obtained in Aβ(12-40).In contrast the shortened Aβ(20-30) peptide showed almost completelyabolished affinity. Mass spectrometric studies of peptides upon H-Dequilibrium exchange showed rapid deuterium incorporation of peptidebackbone hydrogens only for the Aβ sequence (20-30), but only littlebackbone deuteration for residues (30-37), suggesting increasedshielding in this part due to conformational or aggregation effects.Control binding studies of the epitope peptide Aβ(20-37) with antibodiesthat recognized the N-terminal, Aβ(1-16) peptide (plaque-specific mono-and polyclonal antibodies) did not show any binding affinity.

C. Affinity Evaluation of Purified Anti-Aβ(21-37)-Autoantibodies

To assess the quality of the affinity purified antibodies, ELISA wasperformed using the flow through of the affinity-purification column(immobilized Aβ12-40) as a control. The 96-well plate was incubated with200 ng/well of Aβ(1-40) in PBS buffer for 2 hrs at 20° C. The plate waswashed 4 times with 200 μl of PBS containing 0.05% Tween-20 and blockedfor 2 hours with 5% BSA containing 0.05% Tween-20 in PBS buffer. Theplate was then incubated for 2 hrs at 20° C. under gentle shaking withthe affinity-purified antibodies obtained as described in example 2A in5% BSA, 0.05% Tween-20 using the IVIgG flow through as a control. Afterwashing, anti-human horse-radish peroxidase (HRP) conjugated antibodieswere added to the wells and incubated for 1 hr. After adding thesubstrate OPD the optical density was determined at 450 nm. Affinitieswere determined by competitive ELISA, with K_(d)-values ranging betweenabout 8 to about 15×10⁻⁹ M.

D. Electrophoretic Separation for Sequence Determinations ofAnti-Aβ(21-37)-Autoantibodies

Electrophoretic separation and isolation by isoelectric focusing of theanti-Aβ(21-37)-autoantibodies obtained as described in example 2A wascarried out by 1D- and 2D-SDS-PAGE. Samples were equilibrated for 30 minin 6 M urea, 30% glycerol, 2% w/v SDS, 0.05 M Tris-HCl (pH 8.8), 1% DTTand a trace of bromophenol blue, then for 30 min in the same solutionexcept that DTT was replaced by 4.5% (w/v) iodoacetamide. Isoelectricfocusing (IEF) was carried out with a Multiphor II horizontalelectrophoresis system (Amersham Pharmacia Biotech) using 17 cmimmobilized pH gradient (IPG) strips (pH range 3-10 linear). Thesecond-dimensional separation was carried out with a Bio-Rad Protean IIxi cell vertical electrophoresis system using 10% SDS-PAGE gels of 1.5mm thickness. The IPG strips were rehydrated overnight in a solutioncontaining about 100 μg lyophilized anti-Aβ(21-37)-autoantibody forCoomassie and 30 μg for silver staining solubilised in 7 M urea, 2 Mthiourea, 4% CHAPS, 0.3% DTT, 2% Servalyt pH 3-10 and a trace ofbromophenol blue. The samples were applied using the in-gel rehydrationmethod. Rehydrated strips containing the sample were run in the firstdimension for about 30 kVh at 20° C.

Strips placed on the vertical gels were overlayed with 1% agarose in SDSrunning buffer (25 mM Tris-HCl, 192 mM glycine and 0.1% w/v SDS) andsubjected to electrophoresis at 25 mA/gel for 30 min and 40 mA/gel untilthe tacking dye reached the anodic end of gels. After separation inSDS-PAGE gels, the proteins were visualized by silver staining or bysensitive colloidal Coomassie staining and scanned using a GS-710Calibrated Imaging Densitometer (Bio-Rad) (see FIGS. 12 and 13).

Heavy and Light Chain Isolation

Isolation of light chains and heavy chains of antibodies was made using1D gel electrophoresis. The samples (50-200 μg) were dissolved in samplebuffer (4% SDS, 25% glycerol, 50 mM Tris-buffer, 0.02% Coomassie-blue, 6M urea, pH 6.8) with repeated agitation, sonication and centrifugationto ensure maximum solubilization of the antibodies. Reduction of thedisulfide bridges was performed by reaction with dithiothreitol (DTT) ata 1000-x molar excess for 90 min at 20° C. Subsequently, alkylation ofreduced cysteinyl-sulfhydryl groups was performed by reaction with a 3-xmolar excess of iodoacetamide (IAA)/DTT concentration for 60 min at 20°C. 1D-SDS-PAGE isolation of heavy and light chain bands was performed ona 12% acrylamide gel, using a BIO-RAD Protean-(II) Electrophoresis cell,by application of approximately 20 μg antibody per band. The PDQuestsoftware from Bio-Rad was employed for imaging and analyzing 1-D and 2-Dgels. After the gels had been stained and scanned, separate algorithmsof the PDQuest software were used to reduce background noise levels, gelartifacts, and horizontal or vertical streaking from the image. ThePDQuest software was then used to automatically detect the protein spotsseparated on 2-D gels, and for comparison of different gels.Approximately 20 bands were detected as discerned spots, of which 16heavy chain and 15 light chain spots were analysed and identified bymass spectrometric analysis.

E. Overview of Analytical Strategy and Methods for SequenceDetermination of Anti-Aβ(21-37)-Autoantibodies

The primary structure determinations of antibodies, encompassing aminoacid sequences of heavy and light chains, determination and multiplicityof CDR sequences, disulfide linkages, and N-terminal sequences and theirvariations were performed by a combination of the followingcomplementary and partially overlapping methods (overview in FIG. 10):

(i) Direct Edman N-terminal sequence determinations of intact proteins,heavy and light chains following 1D-electrophoretic isolation andblotting of antibody bands (N-terminus and CDR1 variable domains);(ii) 1D-Electrophoretic separation of heavy and light chains, followedby tryptic digestion of isolated protein bands, HPLC isolation oftryptic peptides, and Edman sequence determinations of peptide fragments(variable sequences and CDR regions);(iii) LC-ESI-MS/MS sequence determinations of proteolytic peptidesisolated by HPLC, using a combination of de-novo sequencing and sequencedetermination from NCBI data base search procedures (variable sequencesand CDR regions);(iv) Mass spectrometric sequence assignments of proteolytic digestpeptides from 2D-gel electrophoretic separations of antibody-isoforms,using high resolution MALDI-FTICR-MS in conjunction with databasesearch; by performing two or more data base search procedures (e.g.,Mascot, Profound search engines); constant domains and partiallyvariable sequence domains;(v) Mass spectrometric analysis of HPLC-isolated proteolytic peptides,using MALDI-TOF-MS (constant region sequences); in addition, MALDI-MS(MALDI-TOF and MALDI-FTICR-MS) of proteolytic peptide fragments withoutand with reduction of disulfide bridges was used for assignment andconfirmation of correct disulfide linkages;(vi) assignments of the heavy and light chain connectivities ofantibody-isoforms were performed by MALDI-MS (MALDI-TOF- andMALDI-FTICR-MS) of proteolytic peptide mixtures following 2D-gelelectrophoretic separation, in which the dithiothreitol (DTT) reductionstep was initially omitted, providing intact disulfide-linked antibodiesduring the isoelectric focusing step. Disulfide reduction and alkylationwas then performed in the second electrophoresis step.

F. Detailed Description of Sequences of Anti-Aβ(21-37)-Autoantibodies

Amino acid sequences were determined for anti-Aβ(21-37)-autoantibodiesisolated by affinity-purification from a) serum-IVIgG, and b) serum-IgGsisolated from two healthy adult individuals (m, 30 yrs). Sequencedeterminations for complete antibodies, heavy and light chains,(identified with disulfide-linkages as described above) are shown withassignments of structural details in FIGS. 29, 30 and 32. In theexperimental details depicted in FIGS. 25 to 30, the differentcomplementary methods used for completing the sequence determinations bycomplementary and overlapping partial sequences are shown by differentunderlining codes for (i), Edman N-terminal protein sequencedeterminations; and (ii-v), sequence determinations of proteolyticpeptides isolated by HPLC using Edman sequencing, LC-MS/MS, MALDI-TOF-MSand high resolution MALDI-FTICR-MS, and MALDI-FTICR-MS of proteolyticpeptide mixtures upon 2D-electrophoretic isolation. Furthermore,intra-disulfide linkages of cystinyl residues and heavy chain-lightchain-disulfide linkages identified at Cys-224 (HC-LC), Cys-230 andCys-233 (HC), have been annotated in the heavy chain sequence. Inaddition to direct assignment of sequence positions of cysteineresidues, disulfide linkages were confirmed by mass spectrometricmolecular weight determinations from tryptic, non-reduced peptidemixtures (not shown), and subsequent DTT-reduced peptides, showing fullagreement with homology comparison from predicted assignment ofdisulfide linkages from crystallographic data of a reference IgG1structure (PDB 1IG, Brookhaven protein structure data base).

From the serum-IVIgG antibodies, sequences were identified for 12 heavychain isoforms and 5 light chain isoforms of the respective variableregions; the light chain sequences comprised 4 kappa and 1 lambda chainsequences. For a major portion of the amino acid sequences, sequencedata were corresponding with, and ascertaining each other by at leasttwo complementary, overlapping partial sequence determinations. Allantibody sequences were identified as IgG₁ subclass molecules.

Noteworthy results in the heavy and light chain sequences are theidentification of several single amino acid variations in the constantdomains, each of which was ascertained by several complementing massspectrometric methods and by Edman sequencing of HLC-isolated trypticpeptides. The N-glycosylation site at N-301, was ascertained by Edmansequence determination of the tryptic peptide (297-304) (EEQYNSTYR);this peptide provided a blank in the sequencing cycle-5. AfterN-deglycosylation with PNGaseF, this peptide yielded the sequencedetermination, N-301; furthermore MALDI-FTICR-MS analysis of thedeglycosylated peptide provided identification of the correct molecularmass. In addition a non-glycosylated Fc sequence variation wasidentified by mutation of N301A and by the presence of partiallynon-glycosylated N.

Variable sequences determined for light and heavy chains are summarizedin FIGS. 25 and 26, comprising 12 sequence variants (heavy chain) and 5(light chain). Sequence variations in the heavy chains were considerablymore frequent than for light chains, except for the N-terminal sequenceswhich showed several sequence variations for the light chains butcomplete homogeneity of the heavy chains. N-Terminal sequences weredetermined by a combination of direct Edman sequence analysis ofproteins, Edman sequencing of tryptic N-terminal peptides, and LC-MS/MSsequencing of HPLC-isolated N-terminal peptides. A characteristicfeature is the single uniform heavy chain N-terminal heavy chain (1-20),in contrast to the light chain N-terminal mutations. The N-terminalmutations of the light chains were confirmed by a blast homology searchusing the NCBI data base.

CDR Sequence domains determined for both heavy and light chain regionsare summarized in FIG. 24 a to d. In agreement with mutations identifiedwithin the variable domains, a higher multiplicity was found for theheavy chain sequences with identifications of up to 7 CDR1, CDR2 andCDR3 sequences; while 4 and 5 sequences were determined for light chainCDR1 and CDR2 domains, and two CDR3 sequence variants. Using a stepwiseexamination of heavy and light chain CDRs, all CDR sequences were inagreement matching the Kabat rules (Kabat, E. A., Wu, T. T., Perry, H.M., Gottesman, K. S.& Foeller, C. (1991) Sequences of Proteins ofImmunological Interest (Department of Health and Human Services, PublicHealth Service, National Institutes of Health, Bethesda, Md.) NIH Publ.No 91-3442 5th Ed and R. Kontermann, S. Dübel (eds.), AntibodyEngineering; Springer Lab Manual Series; Springer, Heidelberg 2001;. TheCDR sequences identified enabled the derivation of consensus sequences.

G. Detailed Description of Experimental Procedures for SequenceDeterminations i) Protein Sample Preparation for Sequence Determinations

Anti-Aβ(21-37)-autoantibodies isolated from serum-IVIgG obtained asdescribed in Example 2A were lyophilized and solubilized in denaturationbuffer (6 M Urea; 50 mM Tris, pH=7.5) at a concentration of 1 μg/μL.Reduction of disulfide bridges was performed with DTT at a 1000× molarexcess, for 2 hrs at 30° C. Subsequent alkylation of free thiol groupswas carried out with iodoacetamide at a 3000× molar excess by reactionfor 1 hr at 20° C. in subdued light. The samples were subsequentlylyophilized before separation of heavy and light chains by 1D-gelelectrophoresis.

ii) N-Terminal Edman Protein Sequence Analysis

Automated amino acid sequence analyses were performed with an AppliedBiosystems 494 HT Procise Sequencer attached to a 140C MicrogradientHPLC system, a 785A Programmable Absorbance Detector and a 610A dataanalysis system. All solvents and reagents used were of analyticalultragrade purity (supplied by Applied Biosystems Europe, Darmstadt,Germany).

The following reagents and materials (Applied Biosystems) were employedin all analyses: Blotting buffer: 25 mM Tris-HCl, 192 mM glycine, 0.1%SDS, 20% methanol; PVDF membrane: ProBlott, Applied Biosystems; Filterpapers: GB005 (Schleicher & Schuell); Blotter: PeqLab PerfectBlueTank-Elektroblotter Web M; staining solution: 0.1% Coomassie Blue R-250in 50% methanol; destaining solution: 50% aqueous methanol.

The antibodies were reduced, alkylated and separated by 1D-SDS-PAGE intoheavy and light chain components as described in 4). Immediately afterelectrophoretic separation the fresh gel was equilibrated in transferbuffer for 10 min. The PVDF membrane (10×10 cm, the size of the gel) waswetted in methanol (analytical grade, Nomiapur) for 1 min and thenequilibrated for 20 min in the transfer buffer. Two sheets of filterpaper were cut to the dimensions of the gel (10×10 cm), and filterpapers were soaked in the transfer buffer.

The blotting sandwich was assembled as follows: A wet filter paper wasplaced onto the anode (+) side of the blotting cassette. Theequilibrated PVDF membrane was placed on top of the filter paper. Theequilibrated gel was placed on top of the transfer membrane. The otherwet filter paper was placed on top of the gel. Care was taken not toinclude air bubbles between the sandwich components. The cassette wasthen closed and immersed into the blotting tank.

The blotting was carried out at constant current 1 mA/cm² for 4 hours.After the protein transfer was completed, the PVDF membrane was washedtwice for 15 min with MilliQ water to remove the SDS and glycine. ThePVDF membrane was then washed with methanol for 1 min and then stainedfor 1 min. The stained membrane was washed with destaining solutionuntil the protein spots were clearly visible from the background. Themembrane was then allowed to dry in air. The protein spots were excised,placed in Eppendorf tubes and stored at 4° C. Before sequence analysisthe spots were washed with 100% methanol until complete destaining. Theprotein spots were placed in the sequencer cartridge and sequenced usingthe standard PL PVDF protein method (pulsed liquid sequencing method forPVDF blotted proteins, Applied Biosystems).

iii) Proteolytic Digestion Of Antibodies Following Gel ElectrophoreticSeparation

Heavy and light chains were separated by 1D-gel electrophoresis with 12%separating gel and 5% stacking gel and stained with colloidal CoomassieBlue as described in 4).

For in-gel proteolytic digestion and subsequent HPLC isolation and massspectrometric analysis of the tryptic peptides, the gel bands were cutout and destained by addition of 60% acetonitrile in MilliQ water for 20min at 25° C. After removal of the supernatant and lyophilization of thegel spot to dryness, 1 ml of a solution of 50 mM NH₄HCO₃ was added forrehydration and incubated for 20 min at 25° C. This procedure wasrepeated two times and the final rehydration was performed with theprotease solution (12.5 ng/μl trypsin in 50 mM NH₄HCO₃) at 4° C. for 45min. The gel spots were then incubated for 12 hrs at 37° C. in 1 ml 50mM NH₄HCO₃ and protein fragments were eluted three times with 1 ml 60%acetonitrile in water for 1 hr. The eluates were lyophilised to drynessand solubilised immediately prior to HPLC and MS analysis.

For protein identification/sequence and data base analyses following2D-electrophoresis, sequence determinations by LC-MS/MS and EdmanN-terminal sequence determinations of proteolytic peptides, the gelspots were excised, subjected to dehydration in acetonitrile, andfollowing removal of acetonitrile in vacuo dried in a vacuum centrifuge.Sample preparation for proteolytic digestion was performed as describedabove, by reduction with a volume of 10 mM dithiotreitol (DTT) in 50 mMNH₄HCO₃ sufficient to cover the gel pieces, and protein was performedfor 1 hr at 56° C. After cooling to room temperature, the DTT-containingsolution was replaced with the same volume of a solution of 55 mMiodoacetamide in 50 mM NH₄HCO₃. After 45 min incubation at roomtemperature in the dark with occasional shaking (vortexing), the gelpieces were washed for 10 min with 50-100 μL of 50 mM NH₄HCO₃, anddehydrated again by addition of the same volume of acetonitrile. Theliquid phase was then removed and the gel pieces were completely driedin a vacuum centrifuge.

Excised gel pieces were digested with trypsin either manually orautomatically using a DigestPro 96 robot (Intavis BioanalyticalInstruments, Langenfeld, Germany) according to literature procedures.For manual in-gel-digestion and subsequent mass spectrometric analysis,the spots were excised and destained by addition of 60% acetonitrile inMilliQ water for 20 min at 25° C. After removal of supernatant andlyophilization of the gel spot, a solution of 50 mM NH₄HCO₃ was addedfor rehydration and incubated for 20 min at 25° C. This procedure wasrepeated two times, and final rehydration was then performed for 45 minwith the protease solution (12.5 ng/μl trypsin in 50 mM NH₄HCO₃) at 4°C. The gel spots were incubated for 12 h at 37° C. in 50 mM NH₄HCO₃ andproteolytic peptides were eluted for 3-4 hrs with 60% acetonitrile inwater. The eluates were lyophilized to dryness and dissolved immediatelybefore MALDI-MS analysis in 5 μl acetonitrile/0.1% trifluoroacetic acidin water (2:1).

Automated in-gel-digestion for subsequent mass spectrometric analysiswas performed with a DigestPro 96 robot (Intavis BionalyticalInstruments). The DigestPro 96 is a commercial digest robot systemconsisting of a Gilson 221XL robot, equipped with a module containing atemperature-regulated aluminium reactor block. The block can hold up to96 protein samples and is mounted on rails so that it can be moved bythe robot arm to either a washing or a sample collection position.Protein gel pieces were excised from the 2D-PAGE gels and loaded into aclean 96 well PCR plate which contained small holes pierced into thewell bottoms. The plate was covered by a silicone membrane held in placeby a lid and four mounting screws. The holes in the silicone membraneallow for reagent delivery by a specially designed dispensing needle.This needle has a second, outer channel which delivers nitrogen pressureto the reaction wells. Needle positioning allows either the delivery ofliquid to the vial or the ejection from the reactor by 2.6-bar nitrogenpressure. The entire in-gel digestion process, as described below, wasimplemented on the robot platform, and was controlled by the DigestPro96 software (version 4.02; INTAVIS). Briefly, the gel pieces were washedfour times with 50 μl of 50 mM NH₄HCO₃ and after each step dehydratedwith 100 μl acetonitrile. After the last shrinking step, 50 μl of enzymebuffer (12.5 ng/μl trypsin in 50 mM NH₄HCO₃, pH ˜8) were added to thetubes. The enzyme was drawn into the gel pieces for 30 min.Subsequently, 50 μl solution of 50 mM NH₄HCO₃ were added to cover thegel pieces, and after 6 hrs at 37° C. the peptides were extracted. Thefirst extraction was performed with 50 μl NH₄HCO₃ followed by threeextractions with 50 μl of 10% formic acid; between extractions, the gelpieces were dehydrated with acetonitrile as described above. Thecollected extracts were then dried in a vacuum centrifuge andredissolved immediately before MS analysis, in either 5 μl MALDI-MSsolution (acetonitrile:0.1% trifluoroacetic acid in water, 2:1) or 5 μlESI-MS solution (methanol:water:acetic acid, 50:48:2 (v/v/v)). Forin-gel deglycosylation, the gel pieces were swollen in deglycosylationbuffer, which was prepared by mixing 100 μL of a commercialN-Glycosidase F (PNGase F) preparation (Roche, Mannheim, Germany) with100 μL of 0.1 M ammonium bicarbonate buffer to provide a final enzymeconcentration of 100 units mL⁻¹. If all liquid was taken up by the gelpieces, further digestion buffer (but without PNGase F) was added to thesample to keep the sample wet during overnight incubation at 37° C. Toavoid possible interference from PNGase F-related peptides in MALDI-MSanalyses, the glycosidase was removed prior to proteolysis, by washingthe gel pieces with 0.1% SDS in 0.1 M ammonium bicarbonate (four times250 μL for 1 hr each). All washing solutions were discarded and SDS wasremoved by incubation with 50:45:5 (v/v/v) methanol:water:acetic acid(30 min) and three times washing using 50% acetonitrile in 0.1 Mammonium bicarbonate (30 min each). All washings were discarded, and thegel plugs then dried in a vacuum centrifuge. The same procedure was usedfor in-gel deglycosylation with EndoH glycosidase.

A ZipTip-cleanup procedure was then performed using ZipTip®_(C18)pipette tips from Millipore (Eschborn, Germany). A ZipTip pipette tip isa microcolumn with a resin prepacked into the narrow end of a 10 μlpipette tip. ZipTip pipette tips contain C₁₈ or C₄ reversed-phasematerial for concentrating and purifying peptide and protein samples.ZipTip_(C18) pipette tips were applied for peptides and low molecularweight proteins, while ZipTip_(C4) pipette tips were applied for highermolecular weight proteins. The complete ZipTip procedure was carried outaccording to the instructions of the manufacturer. Briefly, it consistsessentially of five steps: wetting; equilibration of the ZipTip pipettetip; binding of peptides and proteins to the pipette tip; washing; andelution.

iv) HPLC Separation and Isolation of Proteolytic Peptides

All analytical HPLC separations were performed with a BIO-RAD (Muenchen,Germany) 2700 HPLC system using a Vydac C₄ column (250×4.6 mm I.D.) with5 μm silica (300 Å pore size). Linear gradient elution (0 min 0% solventB; 5 min 0% solvent B; 135 min 65% solvent B, 150 min 100% solvent B,160 min 100% B), with eluant A consisting of 0.1% trifluoroacetic acid(TFA) in water, and eluant B of 0.1% TFA in acetonitrile:water (80:20,v/v) at a flow rate of 1 mL/min. The proteolytic peptide samples,typically 50 μg-aliquots were dissolved in 200 μL of eluant A. Detectionof peptides was generally performed at 220 nm using a BIO-RAD variablewavelength absorbance detector.

v) N-terminal Edman Sequence Determinations of Proteolytic Peptides

Tryptic peptides were isolated by HPLC as described above, andlyophilized and stored at −20° C. prior to sequence analysis. The samplesupport used for sequence determinations consisted of a glass fibrefilter (Applied Biosystems) which was treated with a 30 μl., BioBrenePlus (Applied Biosystems) solution (100 μg/μL Biobrene and 6.66 μg/μlNaCl in water), and precycled (3 cycles) using the standard filterprecycle method. For sequence analyses the lyophilized HPLC fractionswere reconstituted in 15 μL 0.1% TFA, containing 20% (v/v) acetonitrilein water. The reconstituted peptide solution was applied on theprecycled glass fiber filter in aliquots of 5 μl to ensure adistribution as close to the centre of the glass fiber filter aspossible, each application followed by drying under a stream of Ar for 1min.

All sequence analyses were performed on an Applied Biosystems 494 HTProcise Sequencer/140C Microgradient System with 785A ProgrammableAbsorbance Detector and 610A Data Analysis System as described above.All solvents and reagents used were from Applied Biosystems. The generalmethod used for the analysis of proteolytic peptides was the standardpulse-liquid method.

vi) Sequence Determinations by ESI-LC-MS/MS of Proteolytic Peptides

All sequence determinations of tryptic peptides isolated by HPLC (seeabove) were performed with a Broker Esquire-3000+ ion-trap LC-MS/MSsystem equipped with nano-ESI/LC ion source systems (Broker Daltonics,Bremen, Germany). HPLC fractions of proteolytic peptides were collectedin 1 ml Eppendorf cups and lyophilized to dryness and stored at −20° C.until LC/MS analysis. The HPLC fractions were dissolved in 16 μl of asolvent mixture containing 1% formic acid in water:acetonitrile (9:1,v/v). The samples were sonicated for 5 min at 20° C. and centrifuged at13 000 rpm/min for 3 min. The content of a sample was transferred into a2 ml screw cap vial equipped with an internal microvial (0.1 ml) andplaced in the LC/MS tray. A 3 μl aliquot of the sample was injected onthe C-18 microcolumn by means of the automatic injection system, and anelution gradient listed in the table below (LC-MS gradient) wasemployed. The sample flows from the injection loop into the column andis then directed into the electrospray interface and through the ionoptics into the ion trap. The total ion current (TIC) was recorded as afunction of time, and is converted into the mass spectrum using the DataAnalysis software (Bruker Daltonics). The most intensive ions to be usedfor MS/MS analysis were selected from the mass spectrum resulting fromthe first LC/MS run. For each precursor ion identified, a separateLC-MS/MS run was performed at identical gradient conditions (see Table).Following start of the pumping system, the isolation and fragmentationof parent ions was switched on in the Esquire Control window, using thespecification of precursor mass, isolation width and fragmentationamplitude. The total ion current (TIC) corresponding to the ionfragments was recorded as a function of time; if a single parent ion wassubjected to fragmentation during the run, the TIC contains a singlepeak. The MS/MS spectrum of the precursor ion was generated by the DataAnalysis software by averaging the pulses at half peak width. The m/zvalues of the fragments contained in the MS/MS spectrum and theirintensities were exported into a data analysis file type (wearing theextension *mgf). The file was uploaded into the MS/MS Mascot searchengine for performing the NCBInr data base search, using the followingsearch parameters: taxonomy, Homo sapiens; allowed missed cleavages, −1;peptide search tolerance, 2 Da; MS/MS tolerance, 0.8 Da; fixedmodification, carbamidomethyl (cysteine); variable modification,Met-oxidation. The results displayed contain the Mowse probability scorein form of a chart, providing the peptide sequence and the proteinoriginating for each hit result. If the result for the fragment ions ofa given precursor led to direct identification score of a peptide fromimmunoglobulin heavy- or light-chain, the peptide sequence obtained wastaken to be a correct one. If no identification score was directlyobtained for a given precursor and its MS/MS spectrum, the peptidesequence was ascertained by de novo sequencing, using the assignmentfunction from the Data Analysis software. This function assigns the massdifference between two fragments into the mass for a specific aminoacid. If the peptide sequence data obtained by the de novo procedure wasidentical with the sequence obtained by the NCBI database search, thesequence result was taken as correct. If the database search performedfor a certain precursor ion did not provide any immunoglobulin peptidefragment, the corresponding precursor ion was assigned as unidentified.

Table of LC-MS/MS gradient elution parameters Time (min) Solvent ASolvent B 0 80 20 3 80 20 6 50 50 16 20 80 18 2 98 20 2 98 22 98 2 24 982vii) MALDI-TOF Mass Spectrometry Of Proteolytic Peptides

MALDI-TOF MS analysis was carried out with a Bruker Biflex linear TOFmass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with anitrogen UV laser (λ=337 nm), a 26-sample SCOUT source, a video systemand a XMASS data system for spectra acquisition and instrument control.A saturated solution of α-cyano-4-hydroxy-cinnamic acid (HCCA) inacetonitrile: 0.1% TFA in water (2:1 v/v) was used as the matrix. Forall MALDI-MS analyses, 0.8 μL of matrix solution and 0.8 μL of thesample solution (proteolytic peptide mixture or tryptic peptidesseparated by HPLC) were mixed on the stainless steel MALDI target andallowed to dry. Acquisition of spectra was carried out at anacceleration voltage (V_(acc)) of 20 kV and a detector voltage of 1.5kV. External calibration was carried out using the average masses ofsingly protonated ion signals of bovine insulin (5734.5 Da), bovineinsulin B-chain oxidized (3496.9), human neurotensin (1673.9 Da), humanangiotensin I (1297.5 Da), human bradykinin (1061.2) and humanangiotensin II (1047.2 Da).

viii) MALDI-FT-ICR-MS of Proteolytic Peptides

MALDI-FTICR mass spectrometric analyses were performed with a BrukerAPEX II FTICR instrument (Bruker Daltonics, Bremen, Germany) equippedwith an actively shielded 7T superconducting magnet (Magnex, Oxford,UK), a cylindrical infinity ICR analyzer cell, and an external Scout 100fully automated X-Y target stage MALDI source with pulsed collision gas.The pulsed nitrogen laser was operated at 337 nm.

Analyses of peptide samples were performed with a 100 mg/mL solution of2,5-dihydroxybenzoic acid (DHB) in acetonitrile/0.1% TFA in water (2:1v/v) used as the matrix. An aliquot of 0.5 μL of matrix solution and 0.5μL of sample solution (tryptic peptide or peptide mixture) were mixed onthe stainless steel MALDI target and allowed to dry. Externalcalibration was carried out using the monoisotopic masses of singlyprotonated ion signals of bovine insulin (5730.609 Da), bovine insulinB-chain oxidized (3494.651), human neurotensin (1672.917 Da), humanangiotensin I (1296.685 Da), human bradykinin (1060.569) and humanangiotensin II (1046.542 Da). Acquisition and processing of spectra wereperformed with XMASS software (Bruker Daltonics, Bremen, Germany).

MALDI-FTICR-MS/MS analyses were performed with the Bruker ApexIIFTICR-MS instrument equipped with SORI-CID(sustained-off-resonance-collision-induced—dissociation) dissociation,IRMPD (Infrared Multiphoton Photodissociation) instrumentation forfragmentation of peptide and protein ions (Damoc et al., 2003). Ionsformed by MALDI ionization were trapped in the analyzer cell, andisolation of a precursor ion was performed by ejecting from the ICR cellall ions of higher and lower masses through the application of suitableexcitation pulses, using the appropriate frequencies and amplitudes. Thefollowing experimental conditions were employed: correlated sweepattenuation: 8-10 dB, ejection safety belt: 500-1000 Hz. For SORI-CID, alow-amplitude rf-excitation was applied for 250 msec to the precursorion at a frequency that is slightly off-resonance (500-1000 Hz) from thecyclotron frequency. The amplitude of the excitation was kept low sothat the ion never went too far from the center of the cell. While thisexcitation was applied, the pressure was raised in the analyzer cell(10⁻⁸ mbar) by admitting a collision gas (argon) through a pulse valvefor 20-80 msec. Under these conditions, the precursor ion underwent manylow-energy collisions, which slowly activated the ion until it reachedits threshold for dissociation.

For IRMPD (infrared-multiphoton-dissociation) experiments themass-selected ions were photodissociated using a 25 W continuous waveCO₂ laser (10.6 μm, Synard, Mukilteo, Wash., USA). The laser power wasset to 50% threshold and the laser irradiation time to 50-200 msec.

For protein identifications and sequence determinations (constant regionsequences) of proteolytic peptide mixtures following 2D-gelelectrophoresis, the following (publicly available) data base searchengines were employed:

Mascot—Peptide mass fingerprint and MS/MS ion search from Matrix ScienceLtd., London.ProFound—Peptide mass fingerprint from Rockefeller and New YorkUniversities.MS-Fit—Peptide mass fingerprint from University of California, SanFrancisco (UCSF).MS-Tag—MS/MS ion search from University of California, San Francisco(UCSF).

Example 3 Determination of Dissociation Constants ofAntigen-Antibody-Complexes by ELISA

The K_(d) values were determined by a modification of the method of Kimet al. (1990). For the determinations, the antibody concentrationsemployed were first derived from an initial calibration curve obtainedby an indirect ELISA as described in example 9B.

1) For the indirect ELISA, microtiter plates were coated with 150μL/well of streptavidin at 20° C. for 2 hrs. Wells were washed one timewith 0.05% (v/v) Tween-20 detergent in phosphate-buffered saline (PBS)(Na₂HPO₄ 5 mM, NaCl 150 mM, pH 7.5). Biotinylated-(G)₅-Aβ (12-40)peptide at concentrations between 1×10⁻⁶ and 10⁻⁸ M were prepared in PBSand deposited in the wells at a volume of 100 μL/well. The wells wereincubated for 2 hours at 20° C. temperature followed by a 4 timeswashing step and blocking with blocking buffer (BSA 5% w/v, 0.05%Tween-20 v/v in PBS) for 2 hours. Anti-Aβ(12-40) antibody was diluted toconcentrations between 1.4×10⁻⁷ and 10⁻⁹ M with blocking buffer andadded at 100 μL/well. The microplate was incubated at 20° C. for 2 hoursand then washed with Covabuffer (0.15 M PBS, pH 7.2 containing 2M NaCl,0.083 M MgSO₄ and 0.05% Tween-20). The wells were incubated withperoxidase-conjugated mouse anti-human IgG (1:5.000) for 45 min at 20°C. Antibody binding was detected with a freshly prepared solution of1,2-Phenylendiamine (OPD) containing 0.1 M citrate-phosphate, 0.1% OPDand 0.006% hydrogen peroxide. The enzyme reaction was monitored as afunction of time at 450 nm, using an ELISA plate reader (Victor², PerkinElmer Life/Analytical Sciences, Boston, Mass.). For each antibody andantigen concentration triplicate wells were prepared and measured.Direct proportionality was observed between absorbance and antibodyconcentration over a wide concentration range. This concentration rangewas used to select the initial concentration for K_(d) determinations.The initial concentration was selected to be within the linear region ofthe plot of optical density vs. antibody concentration.2) For the determination of the K_(d) values the following conditionswere applied. The antigen, Aβ(12-40) peptide at various concentrations(1×10⁻⁶ M to 4.8×10⁻¹⁰ M) was mixed with a constant concentration ofantibody derived from the preliminary ELISA calibration. The incubationwas performed in 5% BSA, 0.05% Tween-20 in PBS using polypropylene testtubes to minimize antibody loss by adsorption on the microreaction tubewalls. After 2 hrs, 100 μl of each mixture was transferred and incubatedfor 30 min into the wells of a microtiter plate previously coated withbiotinylated-(G)₅-Aβ(12-40) (1 μM) and blocked. The concentration offree antibody was then measured by indirect ELISA as described above.The K_(d) values were obtained by plotting the experimental data usingthe Sips coordinates. The following mean K_(d) values were determinedfor the Aβ-antibody complexes of Aβ(12-40) peptide:a) Affinity purified IVIgG antibodies from commercially available IVIgGpreparation:

-   -   8×10⁻⁹M        b) Affinity purified IVIgG antibodies from Serum-A (healthy        human individual; age above 30): 14×10⁻⁹ M        c) Affinity purified IVIgG antibodies from Serum-B (healthy        human individual; age above 30): 18×10⁻⁹ M

Since the range of binding/dissociation constants for the formation ofAβ-fibrils/aggregates has been estimated in the literature to be in therange of 10⁻⁶ M (determined), the binding of antibodies is determined tobe specific. For IgG antibodies, typical K_(d)-values in the range of10⁻⁸ to 10⁻⁹ M have been determined for a large variety of oligo- andpolypeptide antigens and epitopes.

Example 4 Inhibition of Plaque Formation by Affinity Purified IVIgGAntibodies

Human neuroblastoma cells (SH-Sy5y) were grown in RPMI 1640-Mediumsupplemented with 10% fetal calf serum, 10 mM Hepes, 4 mM glutamine andpenicillin (200 units/ml), streptomycin (200 μg/ml). Cells wereincubated at a density of 30,000 cells/well over night in a 96-wellmicrotiter plate. After removal of medium, cells were washed with PBS,and toxic Aβ-oligomers (2 μM final concentration) were added at a volumeof 100 μl fresh medium to 7.5 μM or 15 μM of anti-Aβ(21-37)-autoantibodyor without anti-Aβ(21-37)-autoantibody. The affinity purified IVIgGantibodies were obtained by purifying antibodies from commerciallyavailable IVIgG by affinity chromatography using Aβ(1-40) coupled to agel using the coupling chemistry described in Example 2A. MTT test wasperformed after 4-hrs incubation. FIG. 4 shows that the Aβ-mediatedtoxicity (grey bars) is almost completely antagonized by affinitypurified IVIgGs (black bars).

The experiment was repeated with affinity purified IVIgG (purified asdescribed above, mab CSL Clone 7 (see Example 5). As a negative controlthe antibodies CSL360 (see Example 10) or no antibody was used. Apositive control antibody used was ACA (see Example 10). Results asshown in FIG. 49 clearly show a dose dependent effectiveness ofprotecting cells from the neurotoxic effects of Aβ oligomers for boththe affinity purified mab CSL Clone 7 and affinity purified IVIgG.

Soluble toxic Aβ oligomers as used in Examples 4 can be prepared bydissolving 1.0 mg Aβ in 400 μL HFIP for 10-20 min at room temperature.100 μl of the resulting seedless Aβ solution are then added to 900 μL DDH2O in a siliconized Eppendorf tube. After 10-20 min incubation at roomtemperature, the samples are centrifuged for 15 min. at 14,000×G and thesupernatant fraction (pH 2.8-3.5) is transferred to a new siliconizedtube and subjected to a gentle stream of N2 for 5-10 min to evaporatethe HFIP. The samples are then stirred at 500 RPM using a Teflon coatedmicro stir bar for 24-48 hr at 22° C. Aliquots (10 μl) are taken at 6-12hr intervals for observation by atomic force microscopy or electronmicroscopy.

Example 5 Recombinant Expression of an Anti-Aβ(21-37) Autoantibodies A.Mammalian Expression Vector Construction for Transient Expression

Amino acid sequences for both the light chain variable region of CSLClone 7 (SEQ ID NO: 53) and heavy chain variable region of CSL Clone 7(SEQ ID NO: 60) were used to synthesize cDNA constructs encoding thesesequences by GENEART AG (Regensburg, Germany). The light and heavy chaincDNA constructs were also designed to contain unique flankingrestriction enzyme sites to allow cloning into a mammalian expressionvector upstream of the human light and heavy chain constant regionsrespectively. The constructs were also engineered with a Kozaktranslation initiation sequence, an ATG start codon and signal peptides(MESQTQVLMSLLFWVSGTCG—light chains and MGWSWIFLFLVSGTGGVLS—heavychains).

Using standard molecular biology techniques, the heavy chain variableregion was cloned into the mammalian expression vectorpcDNA3.1(+)-hIgG1, which is based on the pcDNA3.1(+) expression vector(Invitrogen) modified to include the human IgG1 constant region and aterminal stop codon downstream of the variable region insertion site.The light chain variable region was cloned into the expression vectorpcDNA3.1(+)-hκ, which is based on the pcDNA3.1(+) expression vectormodified to include the human kappa constant region and a stop codondownstream of the variable region insertion site.

CSL Clone 7 also was engineered as a “murinized” version to facilitaterepetitive use in murine animal models. The heavy chain variable regionwas cloned into the mammalian expression vector pcDNA3.1(+)-mIgG2a,which is based on the pcDNA3.1(+) expression vector (Invitrogen)modified to include the murine IgG2a constant region and a terminal stopcodon downstream of the variable region insertion site. The light chainvariable region was cloned into the expression vector pcDNA3.1(+)-mκ,which is based on the pcDNA3.1(+) expression vector modified to includethe murine kappa constant region and a stop codon downstream of thevariable region insertion site. Murinized CSL Clone 7 was expressed andpurified as described below.

B. Cell Culture

Serum-free suspension adapted 293-T cells were obtained from GenechoiceInc. Cells were cultured in FreeStyle™ Expression Medium (Invitrogen)supplemented with penicillin/streptomycin/fungizone reagent(Invitrogen). Prior to transfection the cells were maintained at 37° C.in humidified incubators with an atmosphere of 8% CO₂.

C. Transient Transfection

Transient transfection of the clone 7 expression plasmids using 293-Tcells was performed using 293 fectin transfection reagent (Invitrogen)according to the manufacturer's instructions. The light and heavy chainexpression vectors were combined and co-transfected with the 293-Tcells. Cells (1000 ml) were transfected at a final concentration of1×10⁶ viable cells/ml and incubated in a Cellbag 2L (Wave Biotech/GEHealthcare) for 5 days at 37° C. with an atmosphere of 8% CO₂ on a 2/10Wave Bioreactor system 2/10 or 20/50 (Wave Biotech/GE Healthcare). Theculture conditions were 35 rocks per minute with an angle of 8°.Pluronic® F-68 (Invitrogen), to a final concentration of 0.1% v/v, wasadded 4 hours post-transfection. 24 hours post-transfection the cellcultures were supplemented with Tryptone N1 (Organotechnie, France) to afinal concentration of 0.5% v/v. The cell culture supernatants wereharvested by centrifugation at 2500 rpm and were then passed through a0.45 μM filter (Nalgene) prior to purification.

D. Analysis of Protein Expression

After 5 days 20 μl of culture supernatant was electrophoresed on a 4-20%Tris-Glycine SDS polyacrylamide gel and the antibody was visualized bystaining with Coomassie Blue reagent.

E. Antibody Purification

The CSL Clone 7 monoclonal antibody was purified using protein Aaffinity chromatography at 4° C., where MabSelect resin (5 ml, GEHealthcare, UK) was packed into a 30 ml Poly-Prep empty column (Bio-Rad,CA). The resin was first washed with 10 column volumes of pyrogen freeGIBCO Distilled Water (Invitrogen, CA) to remove storage ethanol andthen equilibrated with 5 column volumes of pyrogen free phosphatebuffered saline (PBS) (GIBCO PBS, Invitrogen, CA). The filteredconditioned cell culture media (1 L) was loaded onto the resin bygravity feed. The resin was then washed with 5 column volumes of pyrogenfree PBS to remove non-specific proteins. The bound antibody was elutedwith 2 column volumes of 0.1M glycine pH 2.8 (Sigma, Mo.) into afraction containing 0.2 column volumes of 2M Tris-HCl pH 8.0 (Sigma,Mo.) to neutralize the low pH. The eluted antibody was dialysed for 18hrs at 4° C. in a 12 ml Slide-A-Lyzer cassette MW cutoff 3.5 kD (Pierce,Ill.) against 5 L PBS. The antibody concentration was determined bymeasuring the absorbance at 280 nm using an Ultraspec 3000 (GEHealthcare, UK) spectrophotometer. The purity of the antibody wasanalysed by SDS-PAGE, where 2 μg protein in reducing Sample Buffer(Invitrogen, CA) was loaded onto a Novex 10-20% Tris Glycine Gel(Invitrogen, CA) and a constant voltage of 150V was applied for 90minutes in an XCell SureLock Mini-Cell (Invitrogen, CA) with TrisGlycine SDS running buffer before being visualized using CoomassieStain, as per the manufacturer's instructions.

The above-described techniques can be used to express and purify any ofthe inventive antibodies. In subsequent experiments light chain SEQ IDNOs: 47, 48, 50 to 55, and 145 to 147 were cloned into the expressionvector pcDNA3.1(+)-hκ and co-transfected with heavy chain SEQ ID NOs: 56to 71 and 148 which were cloned into the expression vectorpcDNA3.1(±)-hIgG1. A total of 187 transient transfections were performedcovering all possible light and heavy chain antibody pairs. Thefollowing 42 light and heavy chain antibody pairs (SEQ ID NOs) expressedsufficient antibody for purification and analysis: 47/56, 50/60, 50/61,50/62, 50/67, 50/68, 50/69, 50/148, 51/60, 51/61, 51/62, 51/68, 51/148,52/60, 52/148, 53/60, 53/68, 53/148, 54/60, 54/61, 54/62, 54/67, 54/68,54/69, 54/148, 55/60, 55/61, 55/62, 55/67, 55/68, 55/69, 55/148, 145/60,145/61, 145/62, 145/68, 145/148, 146/60, 146/61, 146/62, 146/68 and146/148.

Such methods can also be employed to produce fully human anti-β amyloidantibodies comprising selected individual sequences based on theconsensus sequences of the respective CDRs, such as SEQ ID NOs: 6 to 11and SEQ ID NOs: 153 to 161, or the single sequences of the respectiveCDRs, such as (a) for CDR I of the heavy chain SEQ ID Nos: 13 to 20, b)for CDR2 of the heavy chain SEQ ID NOs: 21 to 27, c) for CDR3 of theheavy chain SEQ ID NOs: 28 to 32, d) for CDR1 of the light chain SEQ IDNOs: 33 to 37, e) for CDR2 of the light chain SEQ ID NOs: 38 to 43 andSEQ ID NO: 53, f) for CDR3 of the light chain SEQ ID Nos: 44 to 46, g)for the variable heavy chain SEQ ID NOs: 56 to 71 and h) for thevariable light chain SEQ ID NOs: 47 to 55.

For use as a control antibody (ACA) in our studies we also cloned thelight and heavy chain variable region sequences of the humanized 266antibody which is known to bind an epitope contained within position13-28 of the amyloid beta peptide. These sequences were obtained fromthe U.S. Pat. No. 7,195,761 B2. Specifically the genes for the humanizedlight chain variable region of 266 (U.S. Pat. No. 7,195,761 B2, SEQ IDNO: 11) and the humanized heavy chain variable region of 266 (U.S. Pat.No. 7,195,761 B2, SEQ ID No:12) were synthesized, cloned into expressionvectors, transiently expressed and purified using the above-describedmethods.

Example 6 Binding of a Recombinantly ExpressedAnti-Aγ(21-37)-Autoantibody CSL Clone 7 and Affinity Purified IVIgG toOligomeric Forms of Aβ

A synthetic amyloid beta 1-40 peptide (PSL GmbH Heidelberg) containingan additional cysteine at the amino terminal (Aβ1-40.Cys) was analyzedin an immunoprecipitation assay against anti-Aβ(21-37) monoclonalantibody (mab) CSL Clone 7 and against affinity purified IVIgG asdescribed in example 4. PBS was employed as a negative control.

Specifically, it was evaluated whether mab CSL Clone 7 wouldimmunoprecipate the synthetic peptide in a monomer or an oligomer form.The peptide, resuspended (1 mg/ml) in phosphate buffered saline (PBS: 10mM sodium phosphate, 150 mM NaCl, pH 7.4) was used immediately (0 h) orsubjected to oligomerisation (15 h) at 37° C., 900 rpm and stored at−80° C. in small aliquots until use. For the immunoprecipitation,aliquots of 30 μl of Protein-G beads (GE Healthcare) were incubated with5 μg antibody mab CSL Clone 7 or 5 μg anti-Aβ(21-37) autoantibodiespurified according to example 2 (Aβ1-40 column, 2 μg Aβ1-40.Cys and 1.5ml PBS over night at 4° C. Immobilized antibody/peptide were collected.After washing (five times) with PBS, the peptide was eluted by adding 1×non-reducing NuPAGE LDS Sample Buffer (Invitrogen) for 10 min at 95° C.Protein separation was done by electrophoresis on NuPAGE 4-12% Bis-TrisGels (Invitrogen) and western transfer on nitrocellulose membranes bywet blot according to the supplier (Invitrogen). Membranes were blockedwith 1× Roti-Block (Roth) and then successively incubated with the firstantibody, 1:6000 Bam90.1 (anti-Aβ) (Sigma) and secondary antibody, goatanti-mouse HRP conjugated (Pierce). A SuperSignal West Dura ExtendedDuration Substrate (Thermo Scientific/Pierce) was used aschemiluminescent substrate.

The results show that mab CSL Clone 7 (see FIG. 33) and affinitypurified IVIgG (see FIG. 37) bind oligomeric forms of Aβ1-40. Inparticular, mab CSL Clone 7 or affinity purified IVIgG co-incubatedeither with monomeric (0 h) or oligomeric (15 h) forms of Aβ1-40,Cysprecipitated oligomeric forms of the peptide. More results on binding tooligomeric forms of Aβ can be found in Example 12D.

Example 7 Peptide Synthesis

Peptides Biotin-G₅-FAEDVGSNKGA-NH₂ (Biotin-G₅-Aβ20-30) andBiotin-G₅-FAEDVGSNKGAIIGLMVG-NH₂ (Biotin-G₅-Aβ20-37) were synthesized bysolid-phase peptide synthesis (SPPS) on a NovaSyn TGR resin, containinga polystyrene-polyethyleneglycol resin and Rink-amide-linker cleavableunder acidic conditions, according to commercially available materialand published literature procedures. 9-Fluorenylmethoxycarbonyl/t-butyl(Fmoc/tBu) chemistry was used throughout for synthesis using asemi-automated Economy Peptide Synthesizer EPS-221 (ABIMED, Germany).The following side-chain protected amino acid derivatives were used:Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Glu(OtBu)-OH. The synthesis was performed according to thefollowing general protocol: (i) DMF washing (3×1 min), (ii) Fmocdeprotection with 2% DBU, 2% piperidine in DMF (15 min), (iii) DMFwashing (6×1 min), (iv) coupling of 5 equiv of Fmoc amino acid:PyBOP:NMMin DMF (40-60 min), (v) DMF washing (3×1 min). For the synthesis of Aβ(20-37) which has a hydrophobic C-terminal part, double coupling of eachamino acid was employed. The biotinylation of the N-terminus was carriedout on the resin using D-(+)-Biotin. After completion of the syntheses,the peptides were cleaved from the resin using a TFA, triethylsilane anddeionized water mixture (95:2.5:2.5, V/V/V) for 3 h at room temperature.The synthetic peptides were used for example in ELISAs.

Example 8 Amino Acid Sequencing of Aβ(21-37)

Sequence determination of all epitopes either identified (isolated Aβepitopes Aβ(4-10) and Aβ(21-37)) or used (e.g. synthetic Aβ(21-37)) wascarried out by means of

a) Edman sequencing;

b) ESI-Tandem MS/MS-sequencing, and

c) FTICR-MS analysis and fragmentation by means of IRMPD-Fragmentation.

Automated amino acid sequence analysis was performed on an AppliedBiosystems Model 494 Procise Sequencer attached to a Model 140CMicrogradient System, a 785A Programmable Absorbance Detector and a 610AData Analysis System.

All solvents and reagents used were of highest analytical grade purity(Applied Biosystems). The sequencing method used was pulsed liquid.Lyophilized samples were dissolved in 10 μL 0.1% TFA. To assure adistribution as close to the centre of the glass fiber filter aspossible, the sample was applied in aliquots of 2 μL, each applicationfollowed by drying under a stream of argon.

Example 9 ELISAs

ELISAs were used for the determination of

-   (a) plaque-specific anti-Aβ(4-10) antibodies in the anti-Aβ    autoantibodies mixture separated from IVIgG-   (b) binding of anti-Aβ autoantibodies from human serum to Aβ(21-37)    peptide, Aβ(12-40) peptide, and Aβ(1-40) peptide-   (c) binding of anti-Aβ autoantibodies separated from IVIgG and from    individual human sera (AD serum and healthy individuals serum) to    Aβ(1-40) peptide and to Aβ(21-37) epitope peptide-   (d) binding of a recombinantly expressed Aβ(21-37) autoantibody (CSL    Clone 7) to Aβ partial sequences

A. ELISA for Aβ(4-10) Antibodies

In this experiment a standard dilution of the antibody (anti-Aβantibodies isolated from IVIgG using a Cys-Aβ(1-40) antigen column) wasused in combination with 12 serial dilutions of Biotin-G5Aβ(4-10)peptide, used as coating antigen. 96-well ELISA plates were coated with150 μL/well streptavidin solution (c=5 μg/mL in PBS) for 2 hours at roomtemperature. After washing the wells four times with PBS-T (0.05%Tween-20 v/v in PBS, pH=7.5), 100 μL/well of biotinylated epitopepeptides (12 serial dilutions from 50 μM to 0.024 μM in PBS, pH=7.5) wasadded and incubated for 2 hours at room temperature. After that, theplates were washed four times with 200 μL/well PBS-T and thenon-specific adsorption sites were blocked with 5% BSA, 0.05% Tween-20in PBS (200 μL/well, 2 h incubation at RT). Then, 100 μL/well of theanti-Aβ autoantibodies isolated from IVIgG (1:150 dilution prepared in5% BSA, 0.05% Tween-20 in PBS) was added to each well. Thereafter, theplates were incubated at room temperature for two hours and subsequentlywashed six times with PBS-T. 100 μL of peroxidase goat anti-human IgGdiluted 5000 times in 5% BSA, 0.05% Tween-20 were added to each well andthe plates were incubated at room temperature for one hour, then theywere washed three times with PBS-T and once with 0.05 M sodiumphosphate-citrate buffer, pH=5. 100 μL of o-phenylenediaminedihydrochloride (OPD) in substrate buffer (phosphate-citrate) at c=1mg/mL with 2 μL of 30% hydrogen-peroxide per 10 mL of substrate bufferwere added. The absorbance at 450 nm was measured on a Wallac 1420Victor2 ELISA Plate.

B. Antibody Determination in Human Serum by Indirect ELISA

96-well ELISA plates were coated with 100 μL/well of Aβ(1-40) peptide(c=2.5 μg/mL in PBS buffer, pH 7.5) for 2 h at room temperature.Thereafter, the plates were washed four times with 200 μL/well ofwashing buffer (PBS-T; PBS with 0.05% Tween-20) and blocked for 2 h atroom temperature with blocking buffer (5% BSA, 0.1% Tween-20 in PBS).After two times washing with PBS-T, the sera were added at an initialdilution of 1:33.3, then diluted 3 fold serially in blocking buffer andincubated for 2 h at room temperature. Then, the plates were washedeight times with PBS-T and goat anti-human IgG conjugated withhorseradish peroxidase diluted 1:5000 in blocking buffer was added tothe plates and incubated for 1 h at RT. The plates were washed fourtimes with PBS-T and two times with 0.05 M sodium phosphate-citratebuffer, pH=5. 100 μL of o-phenylenediamine dihydrochloride (OPD) insubstrate buffer (phosphate-citrate) at c=1 mg/mL with 2 μL of 30%hydrogen-peroxide per 10 mL of substrate buffer were added. Theabsorbance at 450 nm was measured on a Wallac 1420 Victor2 ELISA Plate.Aβ-antibody quantifications were performed with a 1 μg/μl stocksolution, using a BSA reference curve for calibration using thecommercial protein quantification kit Pierce micro-BCA. The resultsobtained are illustrated in FIG. 8. The percentage given illustrates theAβ-antibody concentrations in IVIgG from two separate ELISAdeterminations. Similar results were obtained with the Aβ(21-37)affinity chromatography. To the contrary, affinity chromatography withAβ(4-10) peptide yielded no detectable amounts of polypeptides bindingto the N-terminal epitope. Consequently, the results obtained withAβ(1-40) are equivalent to those obtained with Aβ(21-37) for healthyindividuals.

C. Binding of Anti-A13 Autoantibodies Isolated from IVIgG and fromIndividual Human serum to Aβ1-40 peptide

96-well ELISA plates were coated with 100 μL/well of Aβ1-40 peptide(c=2.5 μg/mL in PBS buffer, pH 7.5) for 2 h at room temperature.Thereafter, the plates were washed four times with 200 μL/well ofwashing buffer (PBS-T; PBS with 0.05% Tween-20) and blocked for 2 h atroom temperature with blocking buffer (5% BSA in PBS). After washing theplates two times with 200 μL/well of PBS-T, 100 μL/well of the 1^(st)antibody (polyclonal anti-Aβ autoantibodies isolated from IVIgG or fromindividual human serum) (8 serial dilutions prepared in blocking buffer;dilutions from 1:250 to 1:32000) was added and incubated for 2 h at roomtemperature. Then, the plates were washed four times with 200 μL/well ofPBS-T and the 2^(nd) antibody (HRP-goat anti-human IgG; c=1 μg/μL)diluted 2000 times in blocking buffer was added (100 μL/well; 2 hincubation at room temperature). After washing the plates three timeswith 200 μL/well of PBS-T and once with 200 μL/well of citrate-phosphatebuffer, pH=5, 100 μL of o-phenylenediamine dihydrochloride (OPD) insubstrate buffer (phosphate-citrate) at c=1 mg/mL with 2 μL of 30%hydrogen-peroxide per 10 mL of substrate buffer were added. Theabsorbance at 450 nm was measured on a Wallac 1420 Victor² ELISA Plate.

D. Binding of a Recombinantly Expressed Aβ(21-37) Autoantibody (CSLClone 7) to Aβ Partial Sequences

Peptides Biotin-G₅-Aβ(1-40), Biotin-G₅-Aβ(12-40) and Biotin-G₅-Aβ(4-10)were compared for binding to a recombinantly expressed Aβ(21-37)autoantibody (CSL Clone 7) by the following indirect ELISA: 96-wellELISA plates were coated with 150 μL/well streptavidin solution (c=2.5μg/mL in PBS pH7.4) for 2 hours at room temperature. After washing thewells four times with 200 μL/well PBS-T (0.05% Tween-20 v/v in PBS,pH=7.4), 100 μL/well of biotinylated epitope peptides 1 μM in PBS,pH=7.5) were added and incubated for 2 hours at room temperature. Afterthat, the plates were washed four times with 200 μL/well PBS-T and thenon-specific adsorption sites were blocked with 5% BSA, 0.1% Tween-20 inPBS (200 μL/well, over night at RT). Then the plates were washed oncewith 200 μL/well with PBS-T. Then, 8 serial dilutions of CSL Clone 7prepared in 5% BSA, 0.1% Tween-20, 1% DMSO in PBS) were added to thewells. Thereafter, the plates were incubated at room temperature for twohours and subsequently washed six times with PBS-T. 100 μl of peroxidasegoat anti-human IgG diluted 5000 times in 5% BSA, 0.1% Tween-20 wereadded to each well and the plates were incubated at room temperature forone hour, then they were washed three times with 200 μL/well PBS-T andonce with 0.05 M sodium phosphate-citrate buffer, pH=5. 100 μL ofo-phenylenediamine dihydrochloride (OPD) in substrate buffer(phosphate-citrate) at c=1 mg/mL with 2 μL of 30% hydrogen-peroxide per10 mL of substrate buffer were added. The absorbance at 450 nm wasmeasured on a Wallac 1420 Victor² ELISA Plate. Background signals of theassay were measured with antibody dilutions incubated in wells lackingthe biotinylated peptides.

The recombinantly expressed anti-Aβ(21-37) autoantibody CSL clone 7 (seeExample 5) was evaluated as described above using Biotin-G₅-Aβ(1-40),Biotin-G₅-Aβ(12-40) and Biotin-G₅-Aβ(4-10). FIG. 39 shows that CSL clone7 binds to Aβ(1-40) and Aβ(12-40) but not to Aβ(4-10).

Example 10 Prevention of Fibril Formation by the Antibodies of theInvention

1 mg of Aβ1-40 (PSL Heidelberg) was dissolved in a LoBind tube(Eppendorf) with 100 μl trifluoroacetic acid 0.1% (TFA) and incubatedfor 1 hour at room temperature. The solution was diluted with PBS to 1mM Aβ1-40. To 100 μl Aβ fiber formation sample, the antibodies wereadded to a final concentration of 1.3 μM.

The following antibodies were examined:

-   -   affinity purified IVIgG as described in Example 4    -   recombinant antibody CSL Clone 7 (as described in example 5)    -   antibody ACA (U.S. Pat. No. 7,195,761 B2)    -   negative control CSL360 (CSL360 is a chimeric antibody and shows        no binding to Aβ(1-40) when tested using biosensor or ELISA        analysis)

The incubation was carried out overnight at 37° C. on a heating block. A2.5 mM Thioflavin T (THT) solution in Glycine buffer pH 9.2 wasprepared. The 100 μl Aβ fiber formation sample was transferred into ablack −96 well plate (Greiner), and 50 μM THT was added. Thefluorescence (excitation 450 nm emission 490 nm) of the samples wasmeasured after 24 hour with a Tecan InfiniTE M200 plate reader. Onemeasurement represents the average of 25 flashes.

As shown in FIG. 40, all tested Aβ antibodies showed an inhibition offibrilization by about 20% as compared to the negative control.

Example 11 Assay Binding of Sera from an AD Patient and an Age MatchedHealthy Control A: Dot Blot

Dot blots of Aβ_(1-15cys-)and Aβ_(1-40Cys-), (both peptides do have acysteine do have at the N-terminus) freshly resuspended in PBS buffer(sample-0 h) or subjected to oligomerisation (sample-15 h) were appliedon nitrocellulose membrane (0.5 μg/3 μl spot).

The membranes were blocked with Roti-Block (Roth) for 1 h at roomtemperature and then incubated with 10 μg primary antibody (6E10,Bam90.1, IVIG, CSL-7, ACA), an AD-serum (AD1) and a serum (K4) from ahealthy human individual) in 20 ml blocking reagent (RotiBlock) overnight at 4° C. Incubation with secondary antibodies (anti-human HRP:1:100,000; anti-mouse HRP: 1:6,000) was done for 1 h at roomtemperature. A SuperSignal West Dura Extended Duration Substrate (ThermoScientific/Pierce) was used as chemiluminescence substrate according tothe instructions of the manufacturer. The signal on X-ray films wasrecorded for 10 s-5 min.

As shown in FIG. 41 all control antibodies showed specificity to theexpected epitope (6E10=Aβ(1-17), Bam90.1=Aβ(13-28)). Purified antibodiesCSL Clone7, anti Aβ(21-37) autoantibodies (purified according to example4), ACA (see example 5), but also antibodies from the AD serum and thehealthy individual bind to Aβ₁₋₄₀ oligomers (sample 15 h). Only 6E10 andthe AD-serum bound to Aβ 1-15 but not the control sera of the healthypatient.

Also, CSL Clone 7 and Aβ(21-37) autoantibodies purified from IVIgG asdescribed in Example 4 exhibited preferential binding toaggregated/oligomeric forms of Aβ (Aβ(1-40) 15 h as opposed to Aβ 1-40 0h). By comparison, the control antibodies 6E10, Bam90.1 and ACA showedno such preference, binding instead equally to both species of Aβ.

TABLE 1 Summary data of the Dot Blot analysis Affinity se- se- CSL-purified rum rum Peptide 6E10 Bam90.1 Clone 7 IVIgG ACA AD1 K4 Aβ 1-40,0 h ++ ++ (trace) − ++ − − Aβ 1-40, ++ ++ +++ ++ ++ +++ +++ 15 h Aβ1-15, 0 h ++ − − − − ++ − Aβ 1-15, ++ − − − − ++ − 15 h

B: ELISA

IgG from serum samples (AD1 and an age matched healthy human individual,see above in Example 11A) after purification on Protein G (Pierce)according to the instructions of the manufacturer were loaded on anAβ(1-16) affinity column (prepared according to Example 1), washed withPBS and 10 mM sodium phosphate pH 6.8 and eluted with 100 mM Glycine pH2.8.

The eluate was analyzed in an ELISA on Biotin-G₅-Aβ(4-10) coated platesas described in Example 9D.

The result shows a higher titer of Aβ(4-10) antibodies in the serum ofthe AD patient as compared to the signal detected for the control samplefrom an age matched healthy human individual (see FIG. 42).

Results represent an early experiment which suggests, at least for thesingle AD1 serum tested, that Aβ(4-10) autoantibodies are present buteither in low amount, of low affinity, or possible both. If theseresults are verified, they indicate that ultra-sensitive assayprocedures will be required to permit the procedure to become routine.

Example 12 Binding Characteristics of Recombinantly Expressed Aβ(21-37)Autoantibodies in ELISA, Biacore and Western Blot

TABLE 2 Binding characteristics of recombinantly expressed Aβ(21-37)autoantibodies in ELISA and Biacore WESTERN BLOT ANTIBODY EIA BIACOREANALYSIS ACA (# 80) ++++ ++++ Binds to all species 53/60 (# 92) ++++++++ Dimer binding 53/60 (# 93) ++++ ++++ Dimer binding 50/60 + +++ nt50/61 + ++ nt 50/62 − − nt 50/67 − − nt 50/68 − − nt 50/69 − nt nt 50/148 − + nt 47/56 − − nt 51/60 ++++ +++ Dimer binding 51/61 + + nt51/62 − − nt 51/68 − + nt  51/148 + + nt 52/60 ++++ ++++ Dimer binding 52/148 + ++ nt 53/68 − − nt  53/148 ++++ ++++ nt 54/60 ++ +++ nt 54/61++ ++ Dimer binding 54/62 − − nt 54/67 − No capture nt 54/68 − − nt54/69 − No capture nt 55/60 + ++ nt 55/61 +++ +++ Dimer binding 55/62 −− nt 55/67 − No capture nt 55/68 − − nt 55/69 − − nt  55/148 + + nt145/60  +++++ ++++ nt 145/61  − − nt 145/62  +++ + nt 145/68  − − nt145/148 ++ ++ nt 146/60  ++++ ++++ nt 146/61  ++ ++ nt 146/62  + − nt146/68  +/? − nt 146/148 + +++ nt  54/148 ++ + nt +/− qualitativeassessment of ELISA and biosensor binding where increasing (+) indicatesincrease binding titre on ELISA and on biosensor indicates animprovement on either off-rate or on rate that would suggest an antibodywith comparative higher affinity (−) indicates either no binding byantibodies to immobilised antibodies Nt denotes not tested

All transfectants that expressed immunoglobulin efficiently were testedin an ELISA based on binding to amyloid β peptide. Biosensor data isranked on a qualitative affinity binding assessment of the antibody tothe cys-dimer peptide as described below.

Antibodies which are expressed in high quantities and show binding inELISA or Biacore or Western blot are preferred embodiments of theinvention. A failure to express in high quantity or non-binding in anyof the assays described below does not necessarily mean that theseantibodies would not be functional if either expression would beimproved or more sensitive detection methods were be employed.

A: Aβ Peptide Preparation

Lyophilized 1 mg Aβ(1-40) peptide (Sigma) was resuspended in 200 μl1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Sigma), aliquoted into 1.5 mlEppendorf tubes and lyophilized overnight. The aliquoted Aβ(1-40)peptide was resuspended at 1 mg/ml with dimethyl sulphoxide (DMSO, ICN)and stored at 4° C. This material is referred to as Aβ 1-40 monomer.

To oligomerize the Aβ(1-40) peptide the Aβ(1-40) monomer was diluted to0.1 mg/ml in 1×PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl) andincubated for 3 to 6 days at 37° C. and then stored at 4° C., thismaterial is referred to as Aβ(1-40) oligomer.

Aβ(1-40) peptide with an N-terminal cysteine residue (Aβ(1-40 Cys)) wasresuspended from a lyophilized state in ddH2O at 5.9 mg/ml and stored at−80° C. The Aβ(1-40 Cys) peptide was then diluted to 0.1 mg/ml in 1×PBSand stored at 4° C., this material is referred to as Aβ(1-40 Cys)oligomer.

B: Anti-Beta Amyloid Antibody ELISA Protocol

Aβ(1-40) monomer, Aβ(1-40) oligomer, Aβ(1-40 Cys) oligomer, and controlplate using bovine serum albumin (Sigma) or Aprotinin (Sigma) wereimmobilized at 1 μg/ml in PBS (50 μl per well) on a Nunc Maxisorb96-well plate overnight at 4° C. Wells were washed once with 350 μl WashBuffer (1×PBS, 0.05% (v/v) Tween-20 (Sigma)). Wells were then blockedwith Blocking Buffer (2% (w/v) Difco Skim milk (BD) in PBS, 50-150 μlper well) for 2 hours at room-temperature and washed once with 350 μlWash Buffer.

The primary antibody was serial diluted 1:2 in V-bottom well 96-wellplates (Nunc) at starting concentration of 100 mg/ml in Antibody Buffer(1% (w/v) Bovine Serum Albumin (Sigma) in PBS, 0.05% Tween-20 (Sigma)).

The control antibodies (eg. 6E10 mAb (Sigma) and ACA) were analyzed at astarting concentration of 1 mg/ml

100 μl of primary antibody were transferred in serial dilution to NuncMaxisorb plates and incubated for 2-3 hours at room-temperature. Wellswere washed three times with 350 μl Wash Buffer rapidly.

The secondary antibodies sheep anti-human IgG-HRP and sheep anti-mouseIgG-HRP (Chemicon) were added at 1:1000 in Antibody Buffer (50 μl perwell) and incubated for 30 minutes at room-temperature.

Wells were then washed three times with 350 μl Wash Buffer and theplates developed with TMB Substrate (Millipore, Australia) (50 μl perwell) for 5 minutes. The reaction was stopped with 2M phosphoric acid(25 μl per well) and the plates were read at 450 nm, 0.1 seconds inWallac Victor 2 plate reader.

C: Biosensor Analysis of Aβ(1-40) Interaction with RecombinantlyExpressed Aβ(21-37) Autoantibodies

Peptide Sample Preparation:

Preparation of Peptides for Analysis on Captured Monoclonal Antibodiesis as Described in section “Aβ preparation of peptides” above

Biosensor Immunoglobulin Capture Surface Preparation:

An anti-human immunoglobulin biosensor chip was prepared using a humanantibody capture kit (Biacore, Sweden) as per manufacturer'sinstructions at flow rate of 5 μl/min with a 6 minute contact timeduring NHS/ECD immobilisation. Approximately 10000 resonance units wereachieved on all channels.

Capture Conditions:

All antibodies were captured (25 μg/ml in 0.1 mg/ml BSA, Hepes bufferedsaline) at a flow rate of 20 μl/min for 2 mins. In all experiments flowcell 1 served as a baseline subtracted control channel using human IgG1control antibody (Chemicon, Australia).

The 3 remaining channels were used to capture either control antibody(ACA) or human monoclonal antibodies to test for peptide binding.Peptide binding (60 μl) to the captured antibodies was then analysed ata flow rate of 30 μl/min over all channels simultaneously and allowed todissociate to for 300-600 seconds prior to desorption from the sensorsurface using a 5 μl injection of 3M MgCl₂ as per instructions.

All samples were cooled prior to analysis at 12′C using a Multitemp (GE,Sweden) attached to the biosensor 2000 (GE, Sweden).

A second control experiment was performed for all monoclonal antibodieswhere a 60 μl injection of 0.1 mg/ml BSA, Hepes buffered saline wasdirectly injected post antibody capture to take into accountdissociation of human monoclonal antibody from immobilised captureantibody. This result was manually subtracted from data generated usingpeptides using BIAevaluation software (GE, Sweden).

In all experiments Aβ(1-40 Cys) oligomer was analyzed at 20-50 μg/ml in0.1 mg/ml BSA, Hepes buffered saline. Aβ(1-40) monomer was diluted froma 1 mg/ml stock in 100% DMSO to 10 μg/ml in 0.1 mg/ml BSA, Hepesbuffered saline prior to analysis.

FIGS. 43 to 45 show the results obtained in this analysis. Whereas theACA control antibody show equal binding to both Aβ(1-40) monomer andAβ(1-40 Cys) oligomer most antibodies of the invention show apreferential binding to Aβ(1-40 Cys) oligomer.

D: Anti-Beta Amyloid Antibody Tricine SDS-Page and Western Blot Protocol

Novex Pre-cast 10-20% Tricine gels 10-well (Invitrogen) were loaded with0.5 μg peptide per well in 20 μl (0.5 μg/20 μl=25 μg/ml).

Preparation of molecular weight marker: 20 μl 2× Tricine SampleBuffer—Non-reducing (TSB-NR) (300 mM Tris-HCl, pH 8.45, 24% Glycerol, 8%SDS, 0.005% Coomassie Blue G, 0.005% Phenol Red) were added to 20 μlPre-stained markers (Bio-Rad), and 20 μl per well were used.

Preparation of the Samples:

100 μl 1×TSB-NR loading samples were prepared as follows

Peptides:

Stock Dilution 100 μl Aβ 1-40 1.0 mg/ml 1/40 2.5 μl + 47.5 μl ddH₂O + 50μl 2X monomer TSB-NR Aβ 1-40 0.1 mg/ml ¼ 25 μl + 25 μl ddH₂O + 50 μl 2Xoligomer TSB-NR Aβ 1-40 Cys 0.1 mg/ml ¼ 25 μl + 25 μl ddH₂O + 50 μl 2Xoligomer TSB-NR

Each gel was set up in a 10-lane format as below so that each testantivody was flanked by a molecular weight marker lane and a blankbuffer lane. Molecular weight positions have been marked for simplicity.

1) Pre-stained markers2) Aβ1-40 monomer3) Aβ1-40 oligomer4) Aβ1-40 Cys oligomer

5) 1×TSB-NR

6) Pre-stained markers7) Aβ1-40 monomer8) Aβ1-40 oligomer9) Aβ1-40 Cys oligomer

10) 1×TSB-NR

A further gel was run as above and stained for protein by coomassie anddeep purple. The less sensitive Coomassie shows that the peptide isessentially monomer in lanes 2 and 3 and dimer in lane 4 (or lane 7, 8and 9 respectively which is labeled 1, 2 and 3 in FIGS. 47 and 48). Themore sensitive deep purple reveals a ladder of oligomers in each lane,which is consistent with the staining pattern for mabs 6E10 and ACA.

The Tricine gels were run in XCell SureLock Mini-Cell (Invitrogen) withinner and outer buffer chambers containing Tricine SDS Running Buffer(0.1 M Tris Base, 0.1M Tricine, 0.1% SDS). The electrophoreticseparation was completed applying 125V for 90 minutes.

The gel was then transferred to nitrocellulose as per Membrane FilterPaper Sandwich (Invitrogen) using the XCell II Blot Module (Invitrogen)in the XCell SureLock Mini-Cell (Invitrogen). The inner XCell II BlotModule chamber was filled with chilled 1× Tris-Glycine Transfer Buffer(12 mM Tris Base, 96 mM glycine, 20% methanol)) and the outer chamberwas filled with chilled distilled water.

Transfer was achieved by applying 25V for 1.5 hours and thenitrocellulose membrane was subsequently blocked overnight in 50 mlBlocking Buffer (2% (w/v) Difco Skim milk (BD) in PBS) at 4° C.

The primary antibody, control antibodies 6E10 and ACA were added to themembrane at 0.5 μg/ml and the recombinant Aβ(21-37) antibodies at 10-20μg/ml in 10 ml, 1% (w/v) Bovine Serum Albumin (Sigma) in PBS, 0.05%Tween-20 (Sigma)) and were incubated with shaking for 2-3 hours.Membranes were washed 3 times for 10 minutes with 50 ml Wash Buffer(1×PBS, 0.05% (v/v) Tween-20 (Sigma)). The secondary antibody, sheepanti-human IgG-HRP and sheep anti-mouse IgG-HRP (Chemicon) was added at0.5 μg/ml in 10 ml buffer (as above) and incubated with the membranewith shaking for 30 minutes.

The membranes were washed 3 times for 10 minutes with 50 ml Wash Bufferand subsequently developed with ECL Plus (Perkin-Elmer) on AmershamHyperfilm ECL (GE Lifesciences) (see FIG. 47).

Where the signal was below detection threshold using the above protocol,membranes were washed 3× with Wash buffer and then additionally probedwith biotinylated antihuman IgG1 (Sigma Clone 8c/6-39, 1:2000) for 30-60minute at RT. Membranes were then washed as above. Streptavidinperoxidase (1:4000, Chemicon) was then added for 30 mins and membraneswashed again. This additional step amplified the signal and resulted indetection using ECL substrates as indicated above.

An equivalent gel as probed in the western blot analysis was nottransferred to nitrocellulose. One half was stained with coomassie blueper manufacturers instructions (Novex, Invitrogen) and the other halfwas subjected to deep purple high protein sensitivity staining as permanufacturers instructions (GE, Sweden)

Example 13 Plaque Deposition (Taconic Mice) Antibody Treatment ofTransgenic APP Mice

APP transgenic mice (Tg2576) at 10 month of age received once a week ani.p. injection of 200 μl containing 200 μg (8 g/kg) antibodies for 8weeks. One animal received an affinity purified Aβ autoantibodies fromIVIgG, two animals received a murinized monoclonal antibody againstAβ(CSL Clone 7), and two mice received a negative chimeric monoclonalcontrol antibody (CSL360). At the end of the experimental period,animals were killed by decapitation. The brain was dissected and thehemispheres separated along the midline. One hemisphere was fixed in 4%buffered formaldehyde for 24 h followed by dehydration and paraffinembedding. The other hemisphere was immediately snap-frozen in liquidnitrogen and kept at −80° C.

Plaque Evaluation Materials:

3 μm thick slices of murine brain tissue mounted on microscope slidesfrom Mentzel Glas and immunostained with 6F3D-Antibody (see protocol:Immunohistochemistry).

Transmitting light microscope: Eclipse 80i, containing Plan Apochromatobjectives 2×-40× magnification; Nikon Instruments Europe, Nikon GmbH,Duesseldorf, Germany

Imaging Software: NIS-Elements BR software version 2.3, Nikon; NikonInstruments Europe, Nikon GmbH, Duesseldorf, Germany

Digital sight: 2 Megapixel digital camera, Nikon (Nikon InstrumentsEurope, Nikon GmbH, Duesseldorf, Germany).

Excel Software (Microsoft Office 2003, Microsoft Corp. Redmont, USA.)

Method:

Digital RGB-Pictures were taken using the above-mentioned camera in a40× magnification. A macro was created by recording every step definingthe threshold of intensity, minimum and maximum diameter, excluding e.g.vessels (“objects with holes in the middle”) to ensure equal parametersfor every analysis. The area of interest was defined by applying ameasurement frame. Within this area, the aforementioned criteria wereused to identify “objects” (cluster of pixels) fitting into the schemeprovided by the macro. A binary picture was created by the software forbackground subtraction. Five independent fields per location wereanalyzed (Cortex and Hippocampal formation were evaluated separately).Data of the five analyses were summarized by the software and a smallstatistical analysis was provided, like number of plaques per measuredarea, the quotient of plaque area and measured area as percentage.Detailed data of every object were transmitted into an excel file forfurther analysis.

TABLE 3 Number of Fields 1 Number of Objects 7 Objects per Field 7Measured Area 33152.5 [μm * μm] Objects per Area 0.000211146/[μm * μm]Area Fraction 0.152147 Feature Mean St. Dev Minimum Maximum Area 720.581438.5 0.072697 4222.5 EqDiameter 19.112 23.499 0.30424 73.323 Perimeter106.58 169.48 0.94227 514.37 Width 7.5773 5.9398 0.20097 17.626Circularity 0.70335 0.24485 0.20055 1 MeasuredArea 33152 0 33152 33152

Of special importance is the “Area fraction” as it equals the percentageof plaque area (the marked area) from the measured area (total area).

Measured Features: Area

Area is a principal size criterion. In a non-calibrated system, itexpresses the number of pixels; in a calibrated one, it expresses thereal area (given in μm²).

Area Fraction

Area Fraction is the ratio of the segmented image area and the MeasuredArea (defined as: square unit over selected fields).

Area Fraction=Area/Measured Area

Circularity

Circularity equals “1” only for circles; all other shapes arecharacterized by circularity values smaller than “1”. It is a derivedshape measure, calculated from the area and perimeter. This feature isuseful for examining shape characteristics.

Circularity=4*π*Area/Perimeter²

EqDiameter

The equivalent diameter (EqDiameter) is a size feature derived from thearea. It determines the diameter of a circle with the same area as themeasured object:

EqDiameter=√(4*Area/π)

Object Per Area

Number of objects per square unit over selected fields (measured area),

Perimeter

Perimeter is the total boundary measure. It includes both the outer andinner boundary (if there are holes within an object). The perimeter iscalculated from four projections in the directions 0, 45, 90 and 135degrees using Crofton's formula.

Perimeter=π*(Pr0+Pr45+Pr90+Pr135)/4

Width

Width is a derived feature appropriate for elongated or thin structures.It is based on the rod model and is calculated according to:

Width=Area/Length

Example 14 Evidence that the Antibodies of the Invention do not ShowVessel Staining Methods:

3 μm paraffin slices were cut from post-mortem brain material of apatient suffering from Alzheimer's disease and cerebral amyloidangiopathy (CAA) using the HM 355 S rotary microtome from Microm (MICROMInternational GmbH, Walldorf, Germany) and mounted on SuperFrost Plusmicroscope slides from Menzel-Glaeser (Menzel-Glaeser GmbH & Co. KGBraunschweig, Germany). All protocol steps were performed at roomtemperature if not stated otherwise.

De-paraffining of the microscope slides was performed according to thefollowing protocol: xylene (4 changes), 96% ethanol (3 changes), 70%ethanol (3 changes) followed by 2 changes of de-ionised water. Each stepwas performed for 3 minutes.

As pre-treatment for the antigen retrieval, slides were incubated in 70%(v/v) formic acid in PBS for 20 minutes, replaced by de-ionised waterand two changes of PBS as washing steps. The endogenous peroxidase wasblocked for 30 minutes using 1% (v/v) H2O₂ in Methanol. To preventunspecific staining, slides were incubated for 30 minutes with dilutedgoat serum (according to the Vectastain® Elite ABC Kit instructions) orfor one hour with diluted mouse IgG (according to the Vectastain®M.O.M.-Kit instructions), respectively. The blocking solutions wereremoved, no washing step was used.

After the blocking step, primary antibodies were applied to the slidesin a dilution of 1:100 in Vectastain® Elite ABC Kit diluent or 1:50(6F3D, according to the manufacturer's instructions) in Vectastain®M.O.M.-Kit diluent. Negative controls were carried along consisting ofone slide without any antibody or detection system, two slides withoutprimary antibody, but with the Vectastain® Elite ABC Kit or theVectastain® M.O.M.-Kit secondary system, respectively, and two slideswith clone 53/60 recombinant human anti β-amyloid immunoglobulin or the6F3D antibody, respectively, and without any detection system.

The slides were incubated with the primary antibodies overnight (18 hrs)at 4° C. in a humid chamber. The day after, the slides were washed twicefor 2 minutes with PBS. Afterwards, the slides were incubated for 30minutes with biotinylated anti-mouse antibody (6F3D antibody) orbiotinylated anti-human antibody (diluted according to the Vectastain®Elite ABC Kit or the Vectastain® M.O.M.-Kit instructions). The slideswere washed twice for 2 minutes with PBS. Subsequently, the slides wereincubated for 30 minutes with the Vectastain® Elite ABC reagentaccording to the instructions of the Vectastain® Elite ABC Kit and theVectastain® M.O.M.-Kit. This step was followed by two washing steps withPBS for 2 minutes. Afterwards, the DAB reagent was applied for 5 minutes(according to the manufacturer's instructions) as chromogen. Thisreaction was stopped by a washing step in de-ionised water for 5minutes. A 10 second dip in Mayer's acid haemalaun-solution followed byblueing for 5 minutes in running tap-water served as a counterstain.Dehydration was performed by putting the slides into a sequence of 70%(v/v) ethanol (3 times), 96% ethanol (3 times), isopropanol (once),xylene (4 times) for 30 seconds each. The slides were air-dried andmounted with RotiHistokit® and coverslips from Menzel-Glas. Images weretaken using the Nikon Eclipse 80i microscope with a Nikon digitalsight 2Megapixel camera and the Nikon NIS-Elements BR version 2.3 software.

The results shown in FIGS. 48 a to 48 f demonstrate that neither theaffinity purified autoantibodies (purified as in Example 4) nor mab CSLClone 7 (see Example 5) show staining of the vessel walls, whereas theanti-Aβ antibody ACA (see Example 5) shows a staining of the vesselwalls, comparable to that of the antibody 6F3D which was used as apositive control. Such results suggest that the antibodies of theinvention will not trigger adverse events which are believed to becaused by Aβ antibodies binding to Aβ deposited brain vessels (PfeiferM, et al. Cerebral hemorrhage after passive anti-Abeta immunotherapy.Science 298:1379. Herzig M C, et al (2004): Abeta is targeted to thevasculature in a mouse model of hereditary cerebral hemorrhage withamyloidosis. Nat Neurosci 7:954-960).

1-17. (canceled)
 18. A method of detecting or measuring the progressionof a neurodementing disease in a patient, comprising (A) measuring in asample from said patient an antibody titer against a first Aβ peptide,wherein the first Aβ peptide comprises at least the sequence accordingto Aβ(30-37) and at most the sequence according to Aβ(12-40); (B)measuring in a sample from said patient an antibody titer against asecond Aβ peptide wherein the second Aβ peptide comprises at least thesequence according to Aβ(4-10) and at most the sequence according toAβ(1-20); and (C) comparing the titers from steps (A) and (B).
 19. Themethod of claim 18, wherein said neurodementing disease is selected fromthe group consisting of Alzheimer's disease, Down's syndrome, dementiawith Lewy bodies, fronto-temporal dementia, cerebral amyloid angiopathyand amyloidoses.
 20. The method of claim 18, wherein said neurodementingdisease is Alzheimer's disease.
 21. The method of claim 18, wherein thefirst Aβ peptide comprises at least the sequence according to Aβ(21-37).22. The method of claim 18, further comprising comparing said patienttiters with titers determined for normal and AD patients whereby ahigher titer against the first Aβ peptide correlates with a lower riskof development and/or progression of Alzheimer's disease.
 23. The methodof claim 18, further comprising comparing said patient titers withtiters determined for normal and AD patients whereby a higher titeragainst the first Aβ peptide, relative to the titer against the secondAβ peptide correlates with a lower risk of development and/orprogression of Alzheimer's disease.
 24. The method of claim 18, furthercomprising comparing said patient titers with titers determined fornormal and AD patients whereby a higher titer against the second Aβpeptide correlates with a higher risk of development and/or progressionof Alzheimer's disease.
 25. The method of claim 18, further comprisingcomparing said patient titers with titers determined for normal, and ADpatients whereby a higher titer against the second Aβ peptide, relativeto the titer against the first Aβ peptide, correlates with a higher riskof development and/or progression of Alzheimer's disease.
 26. A methodof detecting or measuring the progression of a neurodementing disease ina patient, comprising A) obtaining a first sample from said patient at agiven time point; B) obtaining a second sample from said patient atlater time point; C) measuring in said first and second samples theantibody titer against an epitope comprising at least Aβ(30-37) and atmost Aβ(12-40); and D) comparing the titers of said first and secondsamples.
 27. A method of detecting or measuring the progression of aneurodementing disease in a patient, comprising A) obtaining a firstsample from said patient at a given time point; B) obtaining a secondsample from said patient at later time point; C) measuring in said firstand second samples the antibody titer against an epitope comprising atleast Aβ(4-10) and at most Aβ(1-20); and D) comparing the titers of saidfirst and second samples.
 28. A method of detecting or measuring theprogression of a neurodementing disease in a patient, comprising A)obtaining a first sample from said patient at a given time point; B)obtaining a second sample from said patient at later time point; C)measuring in said first and second samples the antibody titer against anepitope comprising Aβ(30-37); and D) comparing the titers of said firstand second samples.
 29. (canceled)